Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Volcano plot showing differential gene expression in adipose ECs from HFD-fed mice relative to NC-fed mice fed over 12 weeks; genes are displayed as up-(red) or down-regulated (blue). ( B ) UMAP of murine EC Cav1 expression across vascular clusters. ( C ) Dot plot showing Cav1 expression among ECs of different vascular origins and diet-fed mice. ( D ) Volcano plot showing differential gene expression in human adipose ECs from obese individuals relative to lean individuals; genes are displayed as up-(red) or down-regulated (blue). ( E ) UMAP of human EC CAV1 expression across vascular clusters. ( F ) Dot plot showing human CAV1 expression among ECs of different vascular origins from both lean and obese humans.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Schematic outlining the timeline and diet schedule for Cav1 fl/fl mice. ( B ) gDNA excision gel of the Cav1 second exon from lung. ( C ) qPCR of Cav1 RNA isolated from lung. ( D ) en face immunostaining of adipose arteries. N= 3 (each averaged from 3 fields of view), Scale bar = 20µm, (right) quantification in arbitrary fluorescence units (AFU). ( E ) triglycerides, ( F ) cholesterol, ( G ) LDL, ( H ) HDL, ( I ) fasting non-esterified fatty acids (NEFA) from serum of EC Cre - Cav1 fl/fl and EC Cre + Cav1 fl/fl fed a normal chow (NC), N= 8-10 mice per group. ( J ) Lipid staining (Nile red) in an en face adipose artery quantified (below) using lipid droplet area per nuclei. N=5 mice (average from 3 fields of view each), Scale bar = 10 µm. ( K ) Diagram showing loss of Cav1 inhibits lipid uptake. ( L ) Glucose tolerance test (GTT) area under the curve (AUC) quantification. N = 8-10 mice. P-values listed are from unpaired t-tests.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Isolation, Immunostaining, Fluorescence, Staining

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Western blot from lung, showing loss of Cav1 protein when EC Cre is present, quantification on left is normalized to total protein loading control. Unpaired t-test was used, N = 4-5. ( B ) Adipose artery en face with secondary only staining. ( C ) Adipose tissue cross section stained with Cav1, Scale bar = 50µm. ( D ) Weight of mice (in grams; g) over the course of the 12-week NC or HFD diets of Cav1 fl/fl mice ( E ) Percent weight gain calculated using ((final mass-initial mass/Initial mass)*100 of high fat diet (HFD) and normal chow (NC) mice. ( F ) Epididymal fat mass and food intake ( G ) of EC Cre - (N.=4) and EC Cre+ (N= 9) Cav1 fl/fl mice. ( H ) Glucose tolerance test, blood glucose over time after glucose injection. N= 8-10 mice for all experiments for E,F, and H.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Western Blot, Control, Staining, Injection

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Knockdown of Cav1 RNA shown via qPCR. ( B ) Lipid (Bodipy) staining in human adipose microvascular endothelial cells (HAMECs), quantified in ( C ), N= 4 (with each as an average of 3 fields of view). ( D ) Cholesterol concentration after cyclodextrin treatment (+cylco, 10mM) or control treatment (-cyclo). ( E ) Lipid staining (Bodipy) of HAMECs after cyclodextrin and lipid treatments, quantified in ( F ). ( G ) Lipid (Bodipy) staining and quantification ( H ) of cells treated with vehicle or endocytosis inhibitors (dynosore (80µM) and genistein (200µM)) before lipid treatment. ( I ). Nitrite (left) and nitrate (right) levels in EC Cre - Cav1 fl/fl and EC Cre + Cav1 fl/fl mice, N= 5 mice. ( J ) Nitrite and nitrate levels in NC- and HFD-fed mice at 12 weeks of diet. N = 3 mice per group. 50µM lipids were used for treatments in all cells for 15 minutes. Scale bars = 25µm throughout. P-values listed are from unpaired t-tests. 50µM lipids (12:5µM linoleic acid, 25µM oleic acid, 12.5µM palmitic acid)

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Knockdown, Staining, Concentration Assay, Control

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: HAMECs treated with control (siCntrl) or Cav1 (siCav1) siRNA followed by L-NAME and stained for lipid accumulation (Bodipy) after treatment with 50µM lipids (50µM lipids (12:5µM linoleic acid, 25µM oleic acid, 12.5µM palmitic acid). N=4 (each from an average of 3 fields of view). ( B ) Lipid staining (Nile red) in en face adipose arteries of EC Cre - Cav1 fl/fl and EC Cre + Cav1 fl/fl mice given L-NAME (60mg/dL) in their drinking water. N=7 (each from an average of 3 fields of view). ( C ) Cholesterol and triglyceride concentrations from blood taken before and after EC Cre + Cav1 fl/fl mice were administered L-NAME in their drinking water. N=5-6. P-values listed are from paired t-tests. ( D ) Cholesterol and triglyceride concentrations from mice fed a high fat diet (HFD) and administered either regular drinking water or L-NAME water. ( E ) Cholesterol and triglyceride concentrations from RBC Cre - eNOS fl/fl and RBC Cre + eNOS fl/fl mice. N=6-7. ( F ) Cholesterol and triglyceride concentrations from EC Cre - eNOS fl/fl and EC Cre + eNOS fl/fl mice. N=6. P-values listen for ( D-F ) are from unpaired t-tests.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Caveolin lipid raft fractionation in HAMECs with the cell surface marked with biotin, the Cav1-rich lipid raft marked as fraction 3, and calnexin marking ER fractions. ( B ) Immunostaining of an en face adipose artery showing Cav1 (cyan) and CD36 (magenta) in various planes of view (XY, XZ, YZ). Arrows highlight locations in which Cav1 and CD36 are next to one another. Yellow bars show the location in which the XZ and YZ planes were taken from. Scale bar = 10µm ( C ) Control (Cntrl) vs. siCD36-treated HAMECs treated with lipids (50µM lipids; (12:5µM linoleic acid, 25µM oleic acid, 12.5µM palmitic acid)) (stained with Bodipy) and quantified. Scale bar = 25µm. ( D ) HEK cells transfected with CD36 plasmid or empty vector (control) and subsequently treated with lipids and one of two NO donors: DETA/NO or SNOG. Quantification of lipid uptake (below). Scale bar = 25µm, N=5 distinct experiments. ( E ) Biotin switch assay, with subsequent streptavidin bead pull down. All samples treated with DETA/NO. EV = empty vector, CD36 = CD36 plasmid, -B or +B = without or with biotin added, respectively. ( F ) Adipose artery en face , lipid accumulation quantified N = 4, three images were averaged for each value, SSO is Sulfo-N-Succinimidyl oleate a CD36 inhibitor. Scale bar = 20µm. All P-Values denoted are unpaired t-tests.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Fractionation, Immunostaining, Control, Staining, Transfection, Plasmid Preparation, Biotin Switch Assay

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: Western blot showing loss of CD36 band with siCD36 in HAMECs, below is total protein (B) HEK cells with no transfection (top) and HEK cells transfected with CD36 plasmid (bottom). CD36 plasmid is tagged with mCherry ( C ) Cav1 RNA expression with CD36 knockdown in HAMECs measure via q-PCR. ( D ) HEK cells transfected with CD36 plasmid with mcherry reported. ( E ) HEK cell transfections with CD36 plasmid with subsequent CD36 immunoblot. IP = immunoprecipitation. ( F ) Cav1 and eNOS immunoblots in HAMEC vs. HEK cells. ( G ) Biotin switch assay to detect nitrosation of CD36 using C313/3A and C466/313/3A mutant versions of the CD36 plasmid. -B an +B represent without or with biotin respectively. All samples were treated with DETA/NO. ( H ) Cumulative HEK293T lipid uptake data.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Western Blot, Transfection, Plasmid Preparation, RNA Expression, Knockdown, Immunoprecipitation, Biotin Switch Assay, Mutagenesis

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) Structure of CD36 protein with transmembrane and ectodomains marked. Cysteines present in CD36 are marked in red; (left) cysteines in the ectodomain participate in disulfide bonds, (middle) CD36 with four cytoplasmic cysteines palmitoylated, and (right) CD36 with proposed cysteines nitrosated. ( B ) Caveolin lipid raft fractionation from HAMECs treated with Cav1 siRNA. Cav1-rich lipid rafts appear in fraction three when Cav1 is present . Black box marks a shift in fractions where CD36 is enriched. The cell surface is marked with biotin and Calnexin marks ER fractions. ( C ) Membrane biotinylation with subsequent streptavidin bead pull down in HAMECs. HAMECs were treated with control siRNA (Cntrl) or siCav1 and then half were given L-NAME. ( D ) IP-ABE assay on HEK293T cells transfected with CD36-mcherry. Detection of palmitoylated proteins via western blot. ( E ) HAMECs treated with no plasmid control, WT CD36, or C466/3A mutant plasmid. DAPI denotes nuclei, and magenta marks CD36. Scale bar = 10µm. ( F ) En face adipose arteries from EC Cre - Cav1 fl/fl (top), EC Cre + Cav1 fl/fl (middle), and EC Cre + Cav1 fl/fl + L-NAME (bottom) mice stained for CD36 (magenta) and ER with calnexin (green). Quantification of CD36-Calnexin overlap using Pearson’s correlation coefficient on the right, N= 4 mice, statistics represent one-way ANOVA with multiple comparisons.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Fractionation, Membrane, Control, Transfection, Western Blot, Plasmid Preparation, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Nitrosation of CD36 regulates endothelial function and serum lipids

doi: 10.1101/2024.04.09.588733

Figure Lengend Snippet: ( A ) CD36 expression in control and siCav1 treated cells measured via q-PCR. ( B ) Adipose artery en face stained with Alexa Four 568 only. ( C ) CD36 immunoblot from EC Cre - and EC Cre + Cav1 fl/fl mice and quantified in ( D ) by normalizing to total protein. N = 4 mice.

Article Snippet: All HAMECs used were under passage 10. mRNA knockdown was performed using siRNA; Cav1 (30nM, Origene: SR319567) and CD36 (10nM, Thermo Fisher: s534752).

Techniques: Expressing, Control, Staining, Western Blot

Journal: Autophagy

Article Title: Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).

doi: 10.1080/15548627.2020.1851897

Figure Lengend Snippet: Figure 7. Caveolae-mediated endocytosis is involved in the redistribution of endothelial CLDN5 after hypoxia induction. (A) The localization of CLDN5 and CAV1 in bEnd.3 cells under CoCl2-induced hypoxia treatment was imaged by stimulated emission depletion (STED) microscope. White dotted lines-labeled region showed a surrounding of CLDN5 by CAV1 beneath the endothelial cell membrane (yellow arrows). Caveolae-liked vesicle packaging the aggregated CLDN5 (white arrow) was captured and the higher magnification image was shown in the white square. Scale bar: 1 μm. (B) CAV1 and CLDN5 in bEnd.3 cells under CoCl2-induced hypoxia treatment was imaged by immuno-electronmicroscope (IEM). Immunogold-labeled CAV1 was captured and found on the membrane of caveolae-liked vesicle while immunogold-labeled CLDN5 localized inside of the caveolae-liked vesicle. i and ii, normoxia control; iii and iv, hypoxia-treated group. ii and iv are high magnification scans of the red line-marked regions in i and iii respectively. Scale bars: 100 nm. (C-F) After knock- down of Cav1, the redistribution of membranous CLDN5 into the cytosol of bEnd.3 cells (white arrows in D) was suppressed under CoCl2-induced hypoxic conditions. The integrated optical density (IOD) of membranous or cytosolic CLDN5 was quantified. CoCl2: 200 μmol/L, treated for 12 h; Norm: normoxia; Hyp: hypoxia. sicontrol: monolayer of bEnd.3 was transiently transfected with scrambled negative control siRNA. siCav1: monolayer of bEnd.3 was transiently transfected with Cav1 siRNA. n = 3 images for each experiment. Data were presented as mean ± SEM. P value indicates one-way ANOVA with Dunnett’s multiple comparisons test. **, P < 0.01. N.S: no significance. Scale bar: 10 μm.

Article Snippet: For small interfering RNA transfection, the Cav1 siRNA reagent kit (Origene, SR404266) or scrambled negative control siRNA (Origene, SR30004) was used to transfect the bEnd.3 cells according to the manufacturer’s instructions.

Techniques: Microscopy, Labeling, Membrane, Control, Knockdown, Transfection, Negative Control

Journal: Autophagy

Article Title: Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).

doi: 10.1080/15548627.2020.1851897

Figure Lengend Snippet: Figure 8. Autophagy mediates the degradation of endocytosed CAV1 under hypoxia. (A and B) CoCl2-induced hypoxia caused degradation of CAV1 in monolayer bEnd.3 cells. Blocking of autophagy by CQ significantly inhibited the degradation of CAV1 while enhancing of autophagy by Rapa promoted its degradation. n = 5. P value indicates one-way ANOVA with Dunnett’s multiple comparisons test. (C and D) Immunogold-labeled CAV1 (black arrows) was imaged by immuno- electronmicroscope (IEM) and was found in autophagosome-like vesicles of bEnd.3 cells after CoCl2-induced hypoxia treatment. The right panel is a high magnification scan of the red line-marked region in the left panel. The numbers of gold particles which represent packaged CAV1 in caveolae per image were counted for quantification analyses. n = 6 images analyzed. Scale bars: 200 nm. (E) knock-down of Cav1 itself in bEnd.3 cells showed no effect on the paracellular permeability of cell monolayer under normoxic condition. n = 3. CoCl2: 200 μmol/L, treated for 12 h. Norm: normoxia; Hyp: hypoxia; sicontrol: monolayer of bEnd.3 was transiently transfected with scrambled negative control siRNA. siCav1: monolayer of bEnd.3 was transiently transfected with Cav1 siRNA. CQ: chloroquine. Rapa: rapamycin. FITC-dextran: Fluorescein-labeled dextran. Papp: apparent permeability coefficient. LC3: microtubule-associated protein 1 light chain 3. Data were presented as mean ± SEM. P value indicates two-tailed unpaired t test. *, P < 0.05, **, P < 0.01. N.S: no significance.

Article Snippet: For small interfering RNA transfection, the Cav1 siRNA reagent kit (Origene, SR404266) or scrambled negative control siRNA (Origene, SR30004) was used to transfect the bEnd.3 cells according to the manufacturer’s instructions.

Techniques: Blocking Assay, Labeling, Knockdown, Permeability, Transfection, Negative Control, Two Tailed Test

Journal: Autophagy

Article Title: Autophagy alleviates hypoxia-induced blood-brain barrier injury via regulation of CLDN5 (claudin 5).

doi: 10.1080/15548627.2020.1851897

Figure Lengend Snippet: Figure 9. A proposed model of the role of autophagy on protecting the integrity of BBB under hypoxia. Hypoxia treatment in cerebrovascular endothelial cells induces a redistribution of membranous CLDN5 which is further endocytosed by CAV1-composed caveolae, impairing the integrity and permeability of BBB. Meanwhile, endothelial autophagy mediates the clearance of aggregated CLDN5 and CAV1 in the cytosol to reduce cytotoxicity and to block the recycling of CAV1 back to cell membrane, suppressing further redistribution of membranous CLDN5 and preventing BBB from fast disruption. control (CLDN5/LC3B/Nucleus).

Article Snippet: For small interfering RNA transfection, the Cav1 siRNA reagent kit (Origene, SR404266) or scrambled negative control siRNA (Origene, SR30004) was used to transfect the bEnd.3 cells according to the manufacturer’s instructions.

Techniques: Permeability, Blocking Assay, Membrane, Disruption, Control