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16 pages, 1696 KiB

Open AccessArticle

Autologous Splenocyte Reinfusion Improves Antibody-Mediated Immune Response to the 23-Valent Pneumococcal Polysaccharide-Based Vaccine in Splenectomized Mice

by Shengwen Calvin Li and Mustafa H. Kabeer

Biomolecules 2020, 10(5), 704; https://doi.org/10.3390/biom10050704 - 1 May 2020

Cited by 6 | Viewed by 3314

Abstract

Common clinical options, currently, for necessary splenectomy are vaccinations and antibiotic prophylaxis. However, despite these two adjuncts, there still occur numerous cases of overwhelming post-splenectomy infection. To examine whether reperfusion of critical splenic lymphocytes could boost immune response, we harvested splenic lymphocytes, reperfused [...] Read more.

Common clinical options, currently, for necessary splenectomy are vaccinations and antibiotic prophylaxis. However, despite these two adjuncts, there still occur numerous cases of overwhelming post-splenectomy infection. To examine whether reperfusion of critical splenic lymphocytes could boost immune response, we harvested splenic lymphocytes, reperfused the autologous lymphocytes, and then administered a pneumococcal vaccine (PNEUMOVAX^®23, i.e., PPSV23) in splenectomized mice. We found that splenectomy impaired the immune response in the splenectomized group compared to the non-splenectomized group; the splenectomized group with lymphocyte reinfusion had a higher response to polysaccharide vaccination based on antibody titer than the splenectomized group without lymphocyte reinfusion. The sham group with the native spleen had the most elevated antibody titer against the PPSV23 polysaccharide antigen. This may be additive, resulting from contributions of the splenic structure, along with the phagocytic function of the spleen and its constituent cells affecting the antibody response. Reinfusion of splenic lymphocytes may enhance immunity without the complications associated with splenic fragment autotransplantation, which never gained acceptance. This technique is safe and simple since the splenic lymphocytes are autologous and, therefore, not self-reactive, and very similar to autologous blood transfusion. This concept may be beneficial in cases of unavoidable splenectomy, especially in pediatric cases. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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Figure 1
Summary of the procedure for restoring the innate humoral antibody-mediated immunity responsive to Pneumococcal polyvalent 23 serotypes (Pneumovax®23) through autologous splenocyte reinfusion upon splenectomy mice. (A) Flow chart of the procedure—both IgM immune response after initial vaccination (Group A) and IgG immune response following revaccination (Group B). Each group had three separate subgroups (1, 2, and 3). Subgroup 1 (positive control subgroup) were the sham control mice, subgroup 2 (negative control subgroup) consisted of splenectomized mice (−SL), and subgroup 3 (experimental subgroup) consisted of splenectomized mice with autologous splenic lymphocytes (+SL) reinfusion. Inset: micro hematocrit capillary tubes were used to separate serum from blood cells, derived from blood draw via retro-orbital venous plexus. (B) Representatives of FACS analysis of splenic lymphocytes. Fractions of the cells (#062513) were used for FACS analysis with fluorophore-conjugated antibodies against CD3, CD19, CD21, CD35, CD80, respectively, with corresponding isotypes as controls as described in the manufacturer’s Manuel (eBioscience, Affymetrix, Inc., Santa Clara, CA, USA), as described in Methods. Specifically, CD80-FITC (for mature B cells), CD19-PE (pan-marker for global B cells), CD3-eFluor660 (for T cells), CD21-APC (for naïve B cells) were used to determine the purity of cell types of the splenic lymphocyte preparation. As the majority of splenic lymphocytes are B cells and the other B cells are located in lymph nodes and circulation, both CD80 for mature B cells and CD21 for naïve B cells were accessed (Note: Gate R1, 79.4% of the cells were used for analysis regions that illustrated as the analysis histograms). Full article ">Figure 2
Group A mice-humoral antibody-mediated immune response to initial PPSV23 vaccination (A) immediately following splenectomy, as measured by IgM ELISA (B). Y-axis: Absorbance IgM ng/mL, Geo. Mean (i.e., geometric mean) antibody titer (95% CI). X-axis: time interval in a week post PPSV23 vaccination. Antibody IgM titers of Balb/C mice with the initial vaccination and tested against polysaccharides conjugated to ELISA plates. Antibody IgM titers of mice immunized with initial vaccination of PPSV23 polyvalent pneumococcal vaccine, with baseline (i.e., pre-immune) antibody titers not exposed to any pneumococcal antigen. Briefly, humoral immune response (Group A). Group A had three separate subgroups (1, 2, and 3). Subgroup 1 (positive control) was the sham control of 10 mice, subgroup 2 (negative control, splenectomy alone) consisted of 10 splenectomized mice (−SL, i.e., without reinfusion of autologous Splenic Lymphocytes), and subgroup 3 (experimental subgroup) consisted of 10 splenectomized mice (+SL, i.e., with reinfusion of autologous Splenic Lymphocytes). The control animals undergoing a sham laparotomy with spleen intact had the best antibody responses. Splenectomized mice (−SL) had the lowest antibody responses. The subgroup of splenectomized mice (+SL) had an intermediate antibody response. This elevated response in the experimental subgroup (+SL) mice had an immune response as detected by elevated IgM titers in Group A. (* signals the statistically significant, p < 0.001, sky-blue circles (sham control group) compared with red squares (−SL subgroup). # for statistically significant, p < 0.001, grey triangles (+SL) compared with red squares (splenectomy)) (Geo. Mean ± S.D. concentrations (ng/mL titers) and 95% confidence intervals as measured by ELISA with Log10 scale) (“Geo.” stands for geometric mean). Full article ">Figure 3
Group B mice; a humoral antibody-mediated immune response to PPSV23 vaccination (A) immediately following splenectomy, with revaccination at 6 weeks post-splenectomy by IgG ELISA (B). Y-axis: Absorbance IgG ng/mL, Geo. Mean (i.e., geometric mean) antibody titer (95% CI). X-axis: time interval in weeks post PPSV23 vaccination. Antibody IgG titers of Balb/C mice with the repeat vaccination (6 weeks) and tested against polysaccharides conjugated to ELISA plates. Baseline (i.e., pre-immune) antibody titers of mice were not exposed to any pneumococcal antigen. This elevated response in the experimental group (+SL, i.e., with reinfusion of autologous Splenic Lymphocytes) mice had an immune response as detected by elevated IgM titers in Group A (<a href="#biomolecules-10-00704-f002" class="html-fig">Figure 2</a>) and as IgG titers in Group B (<a href="#biomolecules-10-00704-f003" class="html-fig">Figure 3</a>). There was also the mortality of 5% in Group B splenectomized mice (−SL, i.e., without reinfusion of autologous Splenic Lymphocytes) and 40% in Group B splenectomized mice (+SL) near the end of the study period. The Group B splenectomized mice (+SL) had equivalent titers over the long term when monitored out to six weeks as opposed to a low and falling titer observed in the Group B splenectomized mice (without SL). (* signals the statistically significant, p < 0.001, sham control subgroup being compared with −SL subgroup for weeks 10, 11, 12), as sky-blue circles compared with red squares (splenectomy), as shown with mean ± S.D. concentrations (ng/mL titers) and 95% confidence intervals as measured by ELISA with Log10 scale). Doubled-purple-lines defined a time gap from 2 weeks to the repeat vaccination at 6 weeks. (“Geo.”: it stands for geometric mean.). Full article ">Figure 4
Group C mice: Antibody IgG titers were upregulated in mice upon challenges by intravenous injection of inactivated Streptococcus pneumoniae Type III cells as humoral antibody-mediated immune response. Whole Streptococcus pneumoniae Type III cells were inactivated by 0.1% formalin treatment and washed 5x in PBS (pH 7.2) before intravenous injection. Y-axis: Optical density measurement units reflected antibody IgG titers on the ELISA plate reader. X-axis: time interval in a week. The grey line: sham mice possess their spleen and its constituent lymphocytes. The orange line: splenectomized mice (+SL) that did not possess a spleen, but had their splenic lymphocytes reinfused via tail vein injection. The blue line: splenectomized mice (−SL) that did not possess a spleen and did not have any splenic lymphocytes reinfused. Full article ">

11 pages, 2037 KiB

Open AccessArticle

Caveolin-1 Regulates P2Y₂ Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

by Magdiel Martínez, Namyr A. Martínez, Jorge D. Miranda, Héctor M. Maldonado and Walter I. Silva Ortiz

Biomolecules 2019, 9(10), 622; https://doi.org/10.3390/biom9100622 - 18 Oct 2019

Cited by 6 | Viewed by 3598

Abstract

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y₂ receptor (P2Y₂R) to modulate its downstream signaling. P2Y [...] Read more.

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y₂ receptor (P2Y₂R) to modulate its downstream signaling. P2Y₂R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y₂R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y₂R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y₂R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y₂R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y₂R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y₂R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y₂R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y₂R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y₂R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y₂R. These findings support the importance of Cav-1 in modulating P2Y₂R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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shRNA-mediated knockdown of caveolin-1 expression of 1321N1 astrocytoma cells. (A) Immunoblot analysis of hHAP2Y2R, caveolin-1 (Cav-1), and GAPDH (control) expression in serum-starved wild-type (WT) 1321N1 cells (lane 1), human 1321N1 cells expressing hHAP2Y2R (hHAP2Y2R 1321N1 cells) (lane 3), or cells infected with Cav-1 shRNA lentiviral particles (lanes 2 and 4; Cav-1 knockdown (KD)). (B) Densitometric analysis of immunoblots indicates the level of Cav-1 normalized to GAPDH expression. Results are presented as the means ± S.E.M. (n = 3; *** p < 0.001 as determined by one-way ANOVA). Full article ">Figure 2
Cav-1 KD reduced the P2Y2R-mediated ERK1/2 phosphorylation after mechanical injury. Post-injury ERK1/2 phosphorylation time course of (A–C) Cav-1-expressing and (D–F) Cav-1 KD 1321N1 cell lines. Immunoblots for (A) Cav-1-expressing WT-1321N1 and (B) Cav-1/hHAP2Y2R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (C), revealing the post-injury hHAP2Y2R-mediated increased ERK1/2 activity. Immunoblots for (D) Cav-1 KD WT-1321N1 and (E) Cav-1 KD/hHAP2Y2R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (F), revealing the diminished post-injury hHAP2Y2R-mediated increased ERK1/2 activity in Cav-1KD 1321N1 cells. Cells were subjected to a severe traumatic injury and immunoblot analysis was done as described in the Materials and Methods section. ERK1/2 phosphorylation and total ERK1/2, Cav-1, and GAPDH (control) expression in equal amounts of protein were determined by Western blot analysis. Immunoblots are representative of at least three independent experiments. In (C) and (F), ERK1/2 phosphorylation was normalized using the formula: phosphorylated ERK1/2/(total ERK1/2 + GAPDH) and expressed as a percentage of untreated controls at 0 min. Values represent the means ± S.E.M. (n = 4), * p < 0.05 and *** p < 0.001 (one-way ANOVA) represent statistically significant differences between P2Y2R-devoid and P2Y2R-expressing 1321N1 cells. Full article ">Figure 3
Cav-1 KD reduced the P2Y2R-mediated Akt phosphorylation after mechanical injury. Post-injury Akt phosphorylation time course of (A–C) Cav-1-expressing and (D–F) Cav-1 KD 1321N1 cell lines. Immunoblots for (A) Cav-1-expressing WT-1321N1 and (B) Cav-1/hHAP2Y2R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (C), revealing the post-injury hHAP2Y2R-mediated increased Akt activity. Immunoblots for (D) Cav-1 KD WT-1321N1 and (E) Cav-1 KD/hHAP2Y2R-expressing 1321N1 cells. Densitometric analysis of the latter immunoblots is shown in (F), revealing the diminished post-injury hHAP2Y2R-mediated increased Akt activity in Cav-1 KD 1321N1 cells. Cells were lysed and Akt phosphorylation on Ser473 and total Akt, Cav-1, and GAPDH (control) expression in equal amounts of protein were determined by Western blot analysis. Immunoblots are representative of at least three independent experiments. In panels C and F, Akt phosphorylation on Ser473 was normalized using the formula: phosphorylated Akt/(pan Akt + GAPDH) and expressed as a percentage of untreated controls at 0 min. Values represent the means ± S.E.M. (n = 4), where * p < 0.05, ** p < 0.01, and *** p < 0.001 (one-way ANOVA) represent statistically significant differences between P2Y2R-devoid and P2Y2R-expressing 1321N1 cells. Full article ">Figure 4
Cav-1 KD inhibits the (A) P2Y2R-mediated increased cell viability and (B) anti-apoptotic action after mechanical injury. The relative expression of both P2Y2R and Cav-1 are indicated on the x-axes of panels (A) and (B). After injury, cell cultures were returned to the incubator and further incubated for 24 h. Cell viability and caspase-9 activity were measured using AB and caspase-9 fluorometric kit, as described in the Materials and Methods section. Uninjured cells in wells of Flex Plates served as controls. Values are mean ± S.E.M. (n = 4) expressed as a percentage of responses in non-injured cells (upper panel). *** p < 0.001, * p < 0.05 (one-way ANOVA). Full article ">Figure 5
Caveolin-1 is necessary for the P2Y2R anti-apoptotic action in 1321N1 cells after mechanical injury. A schematic representation describing the Cav-1-dependent P2Y2R-mediated signaling pathways investigated in this study. (A) Nucleotide agonists’ activation of the P2Y2R signaling pathways and subsequent antiapoptotic action during mechanical injury. (B) Caveolin-1 knockdown leads to P2Y2R uncoupling from its signaling pathways. Full article ">

16 pages, 2195 KiB

Open AccessEditor’s ChoiceArticle

Caveolin-1 Endows Order in Cholesterol-Rich Detergent Resistant Membranes

by Carla Raggi, Marco Diociaiuti, Giulio Caracciolo, Federica Fratini, Luca Fantozzi, Giovanni Piccaro, Katia Fecchi, Elisabetta Pizzi, Giuseppe Marano, Fiorella Ciaffoni, Elena Bravo, Maria L. Fiani and Massimo Sargiacomo

Biomolecules 2019, 9(7), 287; https://doi.org/10.3390/biom9070287 - 17 Jul 2019

Cited by 12 | Viewed by 4926

Abstract

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on [...] Read more.

Cholesterol-enriched functional portions of plasma membranes, such as caveolae and rafts, were isolated from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice within detergent resistant membranes (DRMs). To gain insight into their molecular composition we performed proteomic and lipid analysis on WT and Cav-1 KO-DRMs that showed predicted variations of proteomic profiles and negligible differences in lipid composition, while Langmuir monolayer technique and small and wide-angle X-ray scattering (SAXS-WAXS) were here originally introduced to study DRMs biophysical association state. Langmuir analysis of Cav-1 containing DRMs displayed an isotherm with a clear-cut feature, suggesting the coexistence of the liquid-ordered (L_o) phase typical of the raft structure, namely “cholesterol-rich L_o phase”, with a phase fully missing in Cav-1 KO that we named “caveolin-induced L_o phase”. Furthermore, while the sole lipid component of both WT and KO-DRMs showed qualitatively similar isotherm configuration, the reinsertion of recombinant Cav-1 into WT-DRMs lipids restored the WT-DRM pattern. X-ray diffraction results confirmed that Cav-1 causes the formation of a “caveolin-induced L_o phase”, as suggested by Langmuir experiments, allowing us to speculate about a possible structural model. These results show that the unique molecular link between Cav-1 and cholesterol can spur functional order in a lipid bilayer strictly derived from biological sources. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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Characterization of detergent-resistant membranes (DRMs)-associated proteins. (a) Equal volumes of sucrose gradient fractions from lungs of wild-type (WT) and caveolin-1 knockout (Cav-1 KO) mice were subjected to western blot analyses for Cav-1, Cav-2, Flot-1, and Cavin-1. (b) Comparative analysis by reverse phase liquid chromatography-tandem mass spectrometry (RP-LC-MS/MS) of DRMs (pooled fractions 3–6). The Venn diagram shows the number of specific and common WT/Cav-1 KO proteins identified in at least two of the three independent biological replicates. (c) Classification of WT and KO DRM proteins into functional categories based on PANTHER v.12. Full article ">Figure 2
Characterization of DRM lipids. Post-nuclear homogenates from WT (a) and Cav-1 KO (b) lung tissue were extracted using 1% TX-100 and fractionated in 5%–30% sucrose gradient. Equal volumes of sucrose gradient fractions were subjected to high performance thin layer chromatography (HPTLC) analysis. Fractions 3–6 corresponding to DRMs showed enrichment of sphingomyelin (SM) and cholesterol (CHOL). (c) HPTLC chromatograms of lipids extracted from DRMs. 10 µg of each lipid standard were spotted on the plate. Cholesterol (CHOL) and triacylglycerols (TG) analysis. Phospholipids (PE; PC) and sphingomyelin (SM) analysis. Lipid concentrations were normalized to DRM protein and calculated against the reference standard. Data are presented as mean ± s.e. (n = 3) (* p < 0.05). Full article ">Figure 3
DRMs compression isotherms. (a) Compression isotherms of Langmuir films prepared with DRMs of WT (red) and Cav-1 KO (black) mice and isotherms relative to the same samples after protein removal (green and blue). (b) Protein-free DRMs isotherms (WT pink and KO blue) in comparison to the isotherm of a simplified “raft” model made of cholesterol, monosialotetrahexosylganglioside (GM1), and dipalmitoylphosphatidylcholine (DPPC) (red) and to the single lipid components (black). (c) The reconstitution of Cav-1 in proteoliposomes: Isotherm obtained after reconstitution (red) in comparison with the KO-DRMs isotherm (black) and the protein-free sample obtained from WT-DRMs, used to prepare proteoliposomes (green). Lo, liquid-ordered phase; Ld, liquid-disordered phase; GM1, monosialotetrahexosylganglioside; DPPC, dipalmitoylphosphatidylcholine. Full article ">Figure 4
X-ray scattering experiments. (a) Small angle X-ray scattering (SAXS) pattern of lung Cav-1 WT-DRMs. The first two Bragg peaks are the first-order reflections of two lamellar phases with d-spacings of d1 = 57.2 and d2 = 39.9 Å. A third diffraction peak, with lamellar periodicity of d3 = 34.0 Å (symbol in read), is also present. (b) The wide-angle X-ray scattering (WAXS) pattern of lung WT-DRMs shows a sharp diffraction peak, characteristic of an ordered lipid phase with a packing spacing of d4 = 4.2 Å. (c) The SAXS pattern of lung KO-DRMs resembled that of Cav-1 WT-DRMs. However, the main difference is the total absence of the diffraction peak d3. (d) Conversely, in the WAXS scan of lung KO-DRMs another contribution appears at d5 = 3 Å, together with the less intense peak relative to d4 = 4.2 Å. Double peak is characteristic of disordered lipid phases. Full article ">Figure 5
Schematic representation of the DRMs bilayer molecular organization. The body of our results, Langmuir isotherm and X-ray scattering experiments, suggest that an ordered phase made of saturated lipids (sphingolipids and gangliosides) and cholesterol is formed (blu), named “cholesterol-rich Lo phase”, together with a more fluid Ld phase made of unsaturated lipids (red). From X-ray scattering results, d1 = 57.2 and d4 = 4.2 Å could be associated with the “cholesterol-rich Lo phase”. A less ordered Ld phase, characterized by d2 = 39.9 and d5 = 3 Å, is also present. Both phases are typical of the lipid-rafts and are present even in the absence of Cav-1 (KO-DRMs). In the presence of Cav-1 a “caveolin-induced Lo phase” can be formed, embedded in the first and characterized by d3 = 34 and d4 = 4.2 Å. Full article ">

Review

Jump to: Research

11 pages, 729 KiB

Open AccessReview

Why Does Hyperuricemia Not Necessarily Induce Gout?

by Wei-Zheng Zhang

Biomolecules 2021, 11(2), 280; https://doi.org/10.3390/biom11020280 - 14 Feb 2021

Cited by 37 | Viewed by 9705

Abstract

Hyperuricemia is a risk factor for gout. It has been well observed that a large proportion of individuals with hyperuricemia have never had a gout flare(s), while some patients with gout can have a normuricemia. This raises a puzzle of the real role [...] Read more.

Hyperuricemia is a risk factor for gout. It has been well observed that a large proportion of individuals with hyperuricemia have never had a gout flare(s), while some patients with gout can have a normuricemia. This raises a puzzle of the real role of serum uric acid (SUA) in the occurrence of gout flares. As the molecule of uric acid has its dual effects in vivo with antioxidant properties as well as being an inflammatory promoter, it has been placed in a delicate position in balancing metabolisms. Gout seems to be a multifactorial metabolic disease and its pathogenesis should not rely solely on hyperuricemia or monosodium urate (MSU) crystals. This critical review aims to unfold the mechanisms of the SUA role participating in gout development. It also discusses some key elements which are prerequisites for the formation of gout in association with the current therapeutic regime. The compilation should be helpful in precisely fighting for a cure of gout clinically and pharmaceutically. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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16 pages, 691 KiB

Open AccessReview

Caveolae and Lipid Rafts in Endothelium: Valuable Organelles for Multiple Functions

by Antonio Filippini and Alessio D’Alessio

Biomolecules 2020, 10(9), 1218; https://doi.org/10.3390/biom10091218 - 21 Aug 2020

Cited by 30 | Viewed by 6217

Abstract

Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and are particularly abundant in endothelial cells and adipocytes. The lipid composition of caveolae largely matches that of lipid rafts microdomains that are particularly enriched in cholesterol, sphingomyelin, glycosphingolipids, and [...] Read more.

Caveolae are flask-shaped invaginations of the plasma membrane found in numerous cell types and are particularly abundant in endothelial cells and adipocytes. The lipid composition of caveolae largely matches that of lipid rafts microdomains that are particularly enriched in cholesterol, sphingomyelin, glycosphingolipids, and saturated fatty acids. Unlike lipid rafts, whose existence remains quite elusive in living cells, caveolae can be clearly distinguished by electron microscope. Despite their similar composition and the sharing of some functions, lipid rafts appear more heterogeneous in terms of size and are more dynamic than caveolae. Following the discovery of caveolin-1, the first molecular marker as well as the unique scaffolding protein of caveolae, we have witnessed a remarkable increase in studies aimed at investigating the role of these organelles in cell functions and human disease. The goal of this review is to discuss the most recent studies related to the role of caveolae and caveolins in endothelial cells. We first recapitulate the major embryological processes leading to the formation of the vascular tree. We next discuss the contribution of caveolins and cavins to membrane biogenesis and cell response to extracellular stimuli. We also address how caveolae and caveolins control endothelial cell metabolism, a central mechanism involved in migration proliferation and angiogenesis. Finally, as regards the emergency caused by COVID-19, we propose to study the caveolar platform as a potential target to block virus entry into endothelial cells. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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18 pages, 3457 KiB

Open AccessReview

Caveolin-3: A Causative Process of Chicken Muscular Dystrophy

by Tateki Kikuchi

Biomolecules 2020, 10(9), 1206; https://doi.org/10.3390/biom10091206 - 20 Aug 2020

Cited by 3 | Viewed by 5336

Abstract

The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, [...] Read more.

The etiology of chicken muscular dystrophy is the synthesis of aberrant WW domain containing E3 ubiquitin-protein ligase 1 (WWP1) protein made by a missense mutation of WWP1 gene. The β-dystroglycan that confers stability to sarcolemma was identified as a substrate of WWP protein, which induces the next molecular collapse. The aberrant WWP1 increases the ubiquitin ligase-mediated ubiquitination following severe degradation of sarcolemmal and cytoplasmic β-dystroglycan, and an erased β-dystroglycan in dystrophic αW fibers will lead to molecular imperfection of the dystrophin-glycoprotein complex (DGC). The DGC is a core protein of costamere that is an essential part of force transduction and protects the muscle fibers from contraction-induced damage. Caveolin-3 (Cav-3) and dystrophin bind competitively to the same site of β-dystroglycan, and excessive Cav-3 on sarcolemma will block the interaction of dystrophin with β-dystroglycan, which is another reason for the disruption of the DGC. It is known that fast-twitch glycolytic fibers are more sensitive and vulnerable to contraction-induced small tears than slow-twitch oxidative fibers under a variety of diseased conditions. Accordingly, the fast glycolytic αW fibers must be easy with rapid damage of sarcolemma corruption seen in chicken muscular dystrophy, but the slow oxidative fibers are able to escape from these damages. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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Chickens with muscular dystrophy (line 413) cannot right themselves from the spine position when they placed on their back while normal birds stand up instantly from this position (a). The pectoralis muscles from normal (b) and dystrophic (c) chickens at seven months are stained with Sirius Red. Normal pectoral muscle fibers (line 412) have polygonal contour and yellow cytoplasm outlined basal lamina by bright red line. They are wrapped by reddish connective tissue. Dystrophic pectoralis muscles are earliest and most severely affected, which are characterized by a marked variation in size with a proliferation of intracellular nuclei, necrotic phagocytosis (arrow), multivesicular fibers (arrow head) and fibrosis with lipid droplets. Note that dystrophic fibers lead to develop thicker endomysium layer compared to age matched wild-type ones. Bars in (b) and (c) indicate 80 and 100 μm, respectively. Full article ">Figure 2
Schematic model indicating the stereographic structure of the relationship among primary myotube (p-myotube), secondary myotube (s-myotube) and myoblasts proliferating within a primary muscle fascicle (a). The s-myotubes take gradually the 2D space on the surface of p-myotubes. They separate from p-myotubes and then occupy the 3D space within a primary muscle fascicle (a) (right). (b) A comparison of myotube formation between complexus and other muscles. Note that a remarkable growth of s-myotubes in complexus muscle loses both 2D and 3D spaces rapidly to develop around p-myotubes compared to other muscles [<a href="#B22-biomolecules-10-01206" class="html-bibr">22</a>,<a href="#B24-biomolecules-10-01206" class="html-bibr">24</a>]. Full article ">Figure 3
Transverse sections stained for the acetylcholinesterase (AChE) activity with histochemical method at motor endplates (m) of: normal (a); heterozygous (b); and dystrophic (c) pectoralis muscles. The AChE activity in normal pectoralis muscle fibers is confined to the motor endplates, while it extends to the extrajunctional sarcoplasm diffusely in heterozygous and dystrophic fibers. The hypertrophied αR fibers are surrounded by atrophied αW fibers (white arrowheads in (b)), some of which have AChE positive endplates (arrowheads in (b)). The majority of dystrophic fibers (c) are hypertrophy and contain intense AChE activity in sarcoplasm and have motor endplates (m), which are stained weaker and thinner than those in other genotypes. Note a positively stained sarcoplasm, likely “Ring fiber”, at the right lower corner (c), Bars = 50 μm. [<a href="#B17-biomolecules-10-01206" class="html-bibr">17</a>]. Full article ">Figure 4
Longitudinal section of succinic dehydrogenase (SDH) activity in: normal (a); dystrophic (b); and combined (normal + dystrophic) donor muscles (c), regenerating in normal host chicks at 56 days post-operation made at 10 days after hatching. The SDH activity in dystrophic fibers is higher than in normal fibers. Compared with homogeneous enzyme reaction in normal and dystrophic donor muscles, SDH activity in combined transplants is more variable along the length, higher (arrowhead) and lower in others (large arrow). Adopted from Kikuchi et al. 1980 [<a href="#B47-biomolecules-10-01206" class="html-bibr">47</a>]. (c) Bar = 100 μm. Full article ">Figure 5
(a) The schematic domain structure of chicken WWP1 and the site of missense mutation. Chicken WWP1 protein is composed of 922 amino acids indicating WWP1 functional domains: C2 domain, three WW domains and HECT domain. C in HECT domain indicates an active cysteine residue. The arrow indicates the site of missense mutation. WW domains bind proline-rich region. (b) Muscle-differentiation markers (Myog, MyoD, MyHC Ia and MyHC IIb) in WWP1-transfected (WT and R436Q) and empty vector-transfected (control) C2C12 cells. Note that the R436Q-transfected cells retained the high expression of both slow MyHC Ia and fast MyHC IIb isoforms compared to control cells. Y-axis indicates relative expression level of each gene to the GAPDH gene expression. Different letters indicate significantly differences (p < 0.05) among column graphs. Adopted from Matsumoto et al. 2008 and 2010 [<a href="#B63-biomolecules-10-01206" class="html-bibr">63</a>]. Full article ">Figure 6
Expression of caveolin-3 (Cav-3) at protein and mRNA level. (a) Expression of Cav-3 in M. pectoralis superficialis (PS), M. anterior latissimus dorsi (ALD) and heart (H) was analyzed by Western blotting. Note that PS expressed higher amount of Cav-3 protein (7.12 ± 3.31-fold) in dystrophic chickens (D), while the expression in ALD and H was undetectable as in normal chickens (N). (b) The semi-quantitative RT-PCR analysis indicated that its mRNA expression was at the similar level between dystrophic (D) and normal (N) pectoralis muscle. Adopted from Matsumoto et al. 2010 [<a href="#B85-biomolecules-10-01206" class="html-bibr">85</a>]. Full article ">

12 pages, 2127 KiB

Open AccessReview

The TGF-β1/p53/PAI-1 Signaling Axis in Vascular Senescence: Role of Caveolin-1

by Rohan Samarakoon, Stephen P. Higgins, Craig E. Higgins and Paul J. Higgins

Biomolecules 2019, 9(8), 341; https://doi.org/10.3390/biom9080341 - 3 Aug 2019

Cited by 49 | Viewed by 9551

Abstract

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, [...] Read more.

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-β1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, arteriosclerosis, and thrombosis, correlating with increased tissue TGF-β1 levels. Additionally, PAI-1 is necessary and sufficient for the induction of p53-dependent replicative senescence. The mechanism of PAI-1 transcription in senescent cells appears to be dependent on caveolin-1 signaling. Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAK^Y577,Y861 and caveolin-1^Y14 phosphorylation are not detected in TGF-β1-stimulated src family kinase (pp60^c-src, Yes, Fyn) triple-deficient (SYF^−/−/−) cells. However, restoration of pp60^c-src expression in SYF-null cells rescued both caveolin-1^Y14 phosphorylation and PAI-1 induction in response to TGF-β1. Furthermore, TGF-β1-initiated Src phosphorylation of caveolin-1^Y14 is critical in Rho-ROCK-mediated suppression of the SMAD phosphatase PPM1A maintaining and, accordingly, SMAD2/3-dependent transcription of the PAI-1 gene. Importantly, TGF-β1 failed to induce PAI-1 expression in caveolin-1-null cells, correlating with reductions in both Rho-GTP loading and SMAD2/3 phosphorylation. These findings implicate caveolin-1 in expression controls on specific TGF-β1/p53 responsive growth arrest genes. Indeed, up-regulation of caveolin-1 appears to stall cells in G₀/G₁ via activation of the p53/p21 cell cycle arrest pathway and restoration of caveolin-1 in caveolin-1-deficient cells rescues TGF-β1 inducibility of the PAI-1 gene. Although the mechanism is unclear, caveolin-1 inhibits p53/MDM2 complex formation resulting in p53 stabilization, induction of p53-target cell cycle arrest genes (including PAI-1), and entrance into premature senescence while stimulating the ATM→p53→p21 pathway. Identification of molecular events underlying senescence-associated PAI-1 expression in response to TGF-β1/src kinase/p53 signaling may provide novel targets for the therapy of cardiovascular disease. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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Comparative structures of lipid rafts and caveolae, two major cholesterol-rich membrane microdomains. Caveolin-1 is a key membrane protein necessary for the formation of cholesterol- and sphingolipid-enriched caveolae. Src family kinases phosphorylate caveolin-1 at Y14 promoting interactions with a subgroup of signaling effectors including the EGFR and FAK, as seen in <a href="#biomolecules-09-00341-f002" class="html-fig">Figure 2</a>. Full article ">Figure 2
A model illustrating the maintenance of induced SMAD3 phosphorylation and PAI-1 transcription in response to TGF-β1 via Src/p53/FAK/Caveolin-1 signaling. Binding of TGF-β1 to the TGF-β receptor (TGF-βR) stimulates the generation of reactive oxygen species (ROS) and activates Src kinases. Src phosphorylates caveolin-1 at Y14 and transactivates the EGFR at the Src target Y845 residue, leading to mobilization of the MEK-ERK and p38 (not shown) pathways. TGF-β1-initiated Src kinase phosphorylation of caveolin-1Y14 also stimulates FAK activation, Rho-GTP loading, and Rho-ROCK activation at sites of integrin/matrix engagement. pCaveolin-1Y14-Rho-ROCK signaling inhibits PTEN-PPM1A interactions, resulting in a reduction of the SMAD phosphatase PPM1A, maintaining pSMAD2/3 levels required for PAI-1 induction and persistent expression in response to TGF-β1. ROS-mediated ATM activation stimulates p53 phosphorylation and recruitment of p-p53 to the promoter region of genes with p53 binding motifs [<a href="#B31-biomolecules-09-00341" class="html-bibr">31</a>,<a href="#B51-biomolecules-09-00341" class="html-bibr">51</a>,<a href="#B53-biomolecules-09-00341" class="html-bibr">53</a>,<a href="#B59-biomolecules-09-00341" class="html-bibr">59</a>]. The PE2 region E box in the PAI-1 promoter is a docking site for the helix-loop-helix transcription-leucine zipper factors USF1/2 which are activated by MAP kinases as well as other TGF-β1-induced kinases. Members of the USF family reorient the DNA minor grove, promoting interactions between SMAD2/3 tethered to the PE2 region SMAD-binding elements (SBEs) with tetramerized p53, bound to its downstream half-site motifs [<a href="#B51-biomolecules-09-00341" class="html-bibr">51</a>,<a href="#B59-biomolecules-09-00341" class="html-bibr">59</a>]. Occupancy of the immediate 5” upstream SMAD-binding elements (SBEs) with SMAD2/3/4 and co-localization with p53, USF2, and the histone acetyltransferases CBP/p300 facilitates the formation of a multi-component transcriptional complex required for TGF-β1-induced PAI-1 expression. Full article ">Figure 3
Signaling requirements for VSMC monolayer scratch would closure. Confluent VSMC cultures were scrape-wounded with a pipette tip prior to addition TGF-β1 or EGF with (+) or without a 30 min pre-incubation with the Rho GTPase inhibitor C3 transferase (C3), the ROCK inhibitor Y-27632, or the EGFR kinase inhibitor AG1478. C3 transferase and Y-27632 effectively attenuated both basal and TGF-β1-stimulated VSMC migration (A) and AG1478 significantly reduced EGF as well as TGF-β1 induced migration (B) in response to monolayer wounding. In TGF-β1-treated cultures, PAI-1 expression was evident at the wound edge within hours post-injury (C, left panel). PAI-1 induction was completely inhibited by exposure to AG1478 (C, right panel) correlating with a significant reduction in cell migration (B). Full article ">

25 pages, 1185 KiB

Open AccessEditor’s ChoiceReview

Cell Intrinsic and Extrinsic Mechanisms of Caveolin-1-Enhanced Metastasis

by America Campos, Renato Burgos-Ravanal, María Fernanda González, Ricardo Huilcaman, Lorena Lobos González and Andrew Frederick Geoffery Quest

Biomolecules 2019, 9(8), 314; https://doi.org/10.3390/biom9080314 - 29 Jul 2019

Cited by 37 | Viewed by 9657

Abstract

Caveolin-1 (CAV1) is a scaffolding protein with a controversial role in cancer. This review will initially discuss earlier studies focused on the role as a tumor suppressor before elaborating subsequently on those relating to function of the protein as a promoter of metastasis. [...] Read more.

Caveolin-1 (CAV1) is a scaffolding protein with a controversial role in cancer. This review will initially discuss earlier studies focused on the role as a tumor suppressor before elaborating subsequently on those relating to function of the protein as a promoter of metastasis. Different mechanisms are summarized illustrating how CAV1 promotes such traits upon expression in cancer cells (intrinsic mechanisms). More recently, it has become apparent that CAV1 is also a secreted protein that can be included into exosomes where it plays a significant role in determining cargo composition. Thus, we will also discuss how CAV1 containing exosomes from metastatic cells promote malignant traits in more benign recipient cells (extrinsic mechanisms). This ability appears, at least in part, attributable to the transfer of specific cargos present due to CAV1 rather than the transfer of CAV1 itself. The evolution of how our perception of CAV1 function has changed since its discovery is summarized graphically in a time line figure. Full article

(This article belongs to the Special Issue Beyond Lipid Rafts and Caveolae: From Caveolae Compartmentalization of Signal Transduction to Medicine)

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