Calciprotein Particles Induce Cellular Compartment-Specific Proteome Alterations in Human Arterial Endothelial Cells

Abstract

Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca²⁺ and PO₄^3- ions in blood, being internalised and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Here, we performed a pathway enrichment analysis of cellular compartment-specific proteomes in primary human coronary artery ECs (HCAEC) and human internal thoracic artery ECs (HITAEC) treated with primary (amorphous) or secondary (crystalline) CPPs (CPP-P and CPPs, respectively). Exposure to CPP-P and CPP-S induced notable upregulation of: (1) cytokine- and chemokine-mediated signaling, Ca²⁺-dependent events, and apoptosis in cytosolic and nuclear proteomes; (2) H⁺ and Ca²⁺ transmembrane transport, generation of reactive oxygen species, mitochondrial outer membrane permeabilisation, and intrinsic apoptosis in the mitochondrial proteome; (3) oxidative, calcium, and endoplasmic reticulum (ER) stress, unfolded protein binding, and apoptosis in the ER proteome. In contrast, transcription, post-transcriptional regulation, translation, cell cycle, and cell-cell adhesion pathways were underrepresented in cytosol and nuclear compartments, whilst biosynthesis of amino acids, mitochondrial translation, fatty acid oxidation, pyruvate dehydrogenase activity, and energy generation were downregulated in the mitochondrial proteome of CPP-treated ECs. Differentially expressed organelle-specific pathways were coherent in HCAEC and HITAEC and between ECs treated with CPP-P or CPP-S. Proteomic analysis of mitochondrial and nuclear lysates from CPP-treated ECs confirmed bioinformatic filtration findings.

Keywords: calciprotein particles; cytosol; endoplasmic reticulum; endothelial cells; lysosomes; mineral stress; mitochondria; molecular signatures; nuclei; proteomic profiling.

Figure 1

Experimental pipeline (A) and bioinformatic analysis of (B) cytosolic, (C) nuclear, (D) mitochondrial, (E) lysosomal, and (F) endoplasmic reticulum (ER) proteins in CPP-P- or CPP-S-treated human coronary artery endothelial cells (HCAEC) as compared to control (PBS-treated) cells. In each letter: principal component analysis (left) demonstrating the relative distance between PBS (sham, blue dots), CPP-P (red triangles), and CPP-S (gray crosses)-treated cells in relation to the proteome of each indicated compartment; Venn diagram (right) showing the number of differentially expressed as well as common proteins in CPP-P vs. PBS, CPP-S vs. PBS, and CPP-P vs. CPP-S comparisons for each of the organelles.

Figure 2

Experimental pipeline (A) and bioinformatic analysis of (B) cytosolic, (C) nuclear, (D) mitochondrial, (E) lysosomal, and (F) endoplasmic reticulum (ER) proteins in CPP-P- or CPP-S-treated human internal thoracic artery endothelial cells (HITAEC) as compared to control (PBS-treated) cells. In each letter: principal component analysis (left) demonstrating the relative distance between PBS (sham, blue dots), CPP-P (red triangles), and CPP-S (gray crosses)-treated cells in relation to the proteome of each indicated compartment; Venn diagram (right) showing the number of differentially expressed as well as common proteins in CPP-P vs. PBS, CPP-S vs. PBS, and CPP-P vs. CPP-S comparisons for each of the organelles.

Figure 3

Bioinformatic analysis of mitochondrial (A) and nuclear (B) lysate in CPP-P- or CPP-S-treated human coronary artery endothelial cells (HCAEC) as compared to control (PBS-treated) cells. In each letter: principal component analysis (left) demonstrating the relative distance between PBS (sham, red triangles), CPP-P (gray crosses), and CPP-S (blue dots)-treated cells in relation to the proteome of each indicated compartment; Venn diagram (right) showing the number of differentially expressed as well as common proteins in CPP-P vs. PBS, CPP-S vs. PBS, and CPP-P vs. CPP-S comparisons for each of the organelles.

Figure 4

Western blotting for apoptosis and endothelial differentiation markers in PBS (sham)-, primary CPP (CPP-P), or secondary CPP (CPP-S)-treated primary human coronary artery endothelial cells (HCAEC). (A) CD31 (endothelial-specific transmembrane glycoprotein, red bands at ≈130 kDa), total and cleaved caspase-3 (an executioner caspase mediating apoptosis, green bands at ≈35 kDa for total caspase-3 and at ≈15 kDa for cleaved caspase-3); (B) ERG (endothelial-specific transcription factor, red bands at ≈60 kDa); (C) endothelial nitric oxide synthase (eNOS, an endothelial-specific enzyme catalysing the synthesis of nitric oxide, red bands at ≈73 kDa; (D) glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a loading control, red bands at ≈40 kDa. Three samples per PBS, CPP-P, or CPP-S group have been measured. Note the significant fraction of cleaved caspase-3 in CPP-P and CPP-S groups, loss of CD31 glycoprotein receptor and ERG transcription factor in CPP-P- and CPP-S treated HCAEC, and equal eNOS synthesis. Shown are uncropped blots.

Figure 5

Dot blotting phosphokinase profiling in PBS (sham)-, primary CPP (CPP-P), or secondary CPP (CPP-S)-treated primary human coronary artery endothelial cells (HCAEC). Hypophosphorylated kinases: red: p38α (phosphorylated at T180/Y182 residues), dark blue: c-Jun N-terminal kinases (JNK) 1/2/3 (phosphorylated at T183/Y185 and/or T221/Y223 residues), green: Src-related FGR kinase (phosphorylated at Y412 residue), orange: glycogen synthase kinase (GSK)-3α/β (phosphorylated at S21/S9 residues), light gold: GSK-3β (phosphorylated at S9 residue), aquamarine: lysine deficient protein kinase (WNK) 1 (phosphorylated at T60 residue), violet: phospholipase C gamma 1 (PLC-γ1, phosphorylated at Y783 residue), brown: Yes kinase (phosphorylated at Y426 residue), black: p53 kinase (phosphorylated at S46 residue), yellow: c-Jun kinase (phosphorylated at S63 residue). Hyperphosphorylated kinases: light gray: p70 S6 kinase (phosphorylated at T389 residue), dark gray: Chk-2 kinase (phosphorylated at T68 residue), dark gold: ribosomal S6 kinases (RSK) 1/2 (phosphorylated at S221/S227 residues), light blue: RSK1/2/3 (phosphorylated at S380/S386/S377 residues), fade blue: proline-rich Akt substrate of 40 kDa (PRAS40) kinase (phosphorylated at T246 residue).