Fig 2: AMPA-type glutamate receptors are expressed in human atherosclerotic plaques. (A) Schematic representation of cell-to-cell glutamatergic communication mechanisms mediated by glutamate ionotropic receptor receptors (Gln, Glutamine; Glu, glutamate; GLS, glutaminase; GLUL, glutamine synthetase). (B) Volcano plot visualization of differentially expressed genes between non-atherosclerotic reference arteries vs. carotid atherosclerotic plaques in the BiKE cohort. Fold change is expressed as “log2 (mean expression in carotid atherosclerotic plaques/mean expression in non-atherosclerotic reference arteries).” Differences between groups were analyzed using unpaired Student's t-test. Glutamate metabolism-related genes are shown in blue, glutamate ionotropic receptors subunits are shown in red when statistically differently expressed, in black when not statistically significantly expressed. GLS, Glutaminase; GLUL, Glutamate-ammonia ligase; GRIA1-4, Glutamate receptor, ionotropic, AMPA 1-4; GRID1-2, Glutamate receptor, ionotropic, delta 1-2; GRIK1-5, Glutamate receptor, ionotropic, kainate 1-5; GRIN1, 2A–D, 3A, Glutamate receptor, ionotropic, N-methyl D-aspartate 1, 2A-D, 3A. Missing genes from these families correspond to missing probes in the microarray dataset. (C) Dot plot showing mRNA levels of glutamate turnover-related genes and glutamate ionotropic receptors subunits, described in (B), in atherosclerotic carotid plaques (n = 127, red) and non-atherosclerotic control arteries (n = 10, blue) from the BiKE cohort microarray data. Dots represent log2 mRNA levels. Middle bar indicates the median mRNA expression and error bars represent SD. (D) Section from a human carotid plaque from the BiKE cohort stained with Hematoxylin QS. L, lumen; FC, Fibrous cap. The black square indicates the region of interest for immune-fluorescent staining shown in the following panels. (E) Consecutive histological sections of the human carotid plaque shown in (D) were stained with antibodies against GRIA1, GRIA2, von Willebrand factor (VWF), smooth muscle actin (SMA), and CD68. Fibrous cap regions are shown. Nuclei visualized with DAPI (blue). (F) Higher magnification of consecutive histological sections of a representative human carotid plaque shown in (E) stained with antibodies against GRIA1, SMA, and GRIA2. White arrows indicate co-localization of SMA (green) with GRIA1 or GRIA2 (red).

Fig 3: Pra-C attenuated increased cortical neuronal excitability by inhibiting cytokines released from microglia. Elevated levels of TNF-a (a) and IL-1ß (b) released from BV-2 cells after LPS stimulation were significantly abolished by pretreatment with Pra-C. (c) Representative results of Western blot analysis showed expression levels of GluN2A, GluN2B, GluA1, GluA2 in primary cultured cortical neurons under different treatment. (d) GluN2A levels didn’t show significant change among these groups. (e–g) MCM from BV-2 activated by LPS with pretreatment with Pra-C notably prevented upregulation of GluN2B (e) and GluA1 (f) in cortical neurons, but no significant change in GluA2 levels. None treated or Pra-C treated MCM, and Pra-C directly incubation didn’t change these excitatory synaptic proteins in cortical neurons (d–g). Each value represents the mean ± SEM of three independent experiments (n = 5 in each group of ELISA assay and n = 4 in each group of Western blot, *p < 0.05, **p < 0.01 vs. control group, #p < 0.05, ##p < 0.01 vs. LPS stimulated group). LPS: Lipopolysaccharides; MCM: microglial conditional medium.

Fig 4: A Comprehensive Analysis of nervous system-associated Transcripts in Carotid Atherosclerosis Segregates Glutaminase, Glutamate-Ammonia Ligase, and AMPA-type Receptors. (A) Pie chart showing classification of the compiled 217 neuronal-associated genes investigated by expression analysis in BiKE plaques. (B) Volcano plot visualization of differentially expressed genes between symptomatic (n = 87) and asymptomatic (n = 40) patient groups. Glutamate-related genes are shown in red when their expression is statistically significantly different between conditions, otherwise glutamate-related markers are represented in black. GRIA1, Glutamate Ionotropic Receptor AMPA-type Subunit 1; GRIA2, Glutamate Ionotropic Receptor AMPA-type Subunit 2; GLS, Glutaminase; GLUL, Glutamate-ammonia ligase. Fold change is expressed as “log2 (mean expression level symptomatic/asymptomatic).” Differences between groups were analyzed using unpaired Student's t-test.

Fig 5: VSMCs express AMPA-type glutamate receptors in atherosclerotic plaques. (A) tSNE visualization of single cell transcriptomic analysis of plaques (n = 5) derived from the right coronary artery of human patients (n = 4), colored according to broad cell clustering as indicated in the figure. Numbers denote unidentified clusters. NK, natural killer; SMC, smooth muscle cell. (B) tSNE visualization of single cell transcriptomic analysis of GLS (red) and GLUL (blue) expression overlaid on the cell clusters from (A). Color legend (right) indicating relative expression levels for GLS1 on x-axis and GLUL on y-axis. (C) tSNE visualization of single cell transcriptomic analysis of GRIA1 (red) and GRIA2 (blue) expression overlaid on the cell clusters from (A). Color legend (right) indicating relative expression levels for GRIA1 on x-axis and GRIA2 on y-axis. (D) Dot plot visualization of the percentage of cells positive for GLS (red) and GLUL (blue) expression within the identified cell clusters. Error bars indicate SD, n = 4 patients. (E) Dot plot visualization of the percentage of cells positive for GRIA1 (red) and GRIA2 (blue) expression within the identified cell clusters. Error bars indicate SD, n = 4 patients.