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Shotgun proteomics analysis of nuclear and mitochondrial from human coronary artery endothelial cells fraction treated with calcioprotein particles

ABSTRACT: Calciprotein particles (CPPs) are indispensable scavengers of excessive Ca2+ and PO43- ions in the blood, being internalized and recycled by liver and spleen macrophages, monocytes, and endothelial cells (ECs). Despite the fact, that CPPs are associated with many cardiovascular diseases and are known to trigger endothelial dysfunction, the exact mechanisms of its recycling by ECs are still poorly studied. Particularly, because of the low resolution of the standard proteomics approach. We assume that CPPs internalization would cause oxidative stress and strong rearrangements in the transcription. Therefore, we performed a shotgun proteomics analysis of nuclear and mitochondrial fraction of human coronary artery endothelial cells (HCAEC) treated with primary (amorphous) or secondary (crystalline) CPPs (CPP-P and CPPs, respectively). Contact: Dr Anton G. Kutikhin, Laboratory of Molecular, Translational and Digital Medicine, Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, Russia (lab head).

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hcaec Cell

SUBMITTER: Egor Repkin

LAB HEAD: Dr. Anton G. Kutikhin

PROVIDER: PXD047581 | Pride | 2023-12-31

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	IKFB_mit_1_Slot1-39_1_3939.d.zip	Other
	IKFB_mit_1_Slot1-39_1_3939.mgf	Mgf
	IKFB_mit_2_Slot1-40_1_3943.d.zip	Other
	IKFB_mit_2_Slot1-40_1_3943.mgf	Mgf
	IKFB_mit_3_Slot1-41_1_3921.d.zip	Other

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Project description:Calciprotein particles (CPPs) represent self-assembling scavengers of excessive Ca2+ and PO43- ions which are formed in the human serum under the guidance of acidic proteins termed mineral chaperones (e.g., fetuin-A and albumin). While protecting the human organism from extraskeletal calcification, circulating CPPs are internalised by Kupffer cells and endothelial cells (ECs) and induce calcium stress upon their dissolution in lysosomes, thereby causing a pro-inflammatory response through the inflammasome-dependent and inflammasome-independent mechanisms. In patients with hyperphosphatemia (e.g., those with end-stage renal disease), a considerable proportion of primary amorphous CPPs transforms into secondary crystallised CPPs which instigate stronger cytokine release and indicate critical depletion of mineral buffering systems. A number of reports documented that serum of patients with chronic kidney disease, arterial hypertension, coronary artery disease, cerebrovascular disease, and autoimmune disorders shows increased CPP generation that reflects uncurbed mineral stress in these clinical scenarios and emphasizes the need in the development of anti-CPP therapies. For the experiments, CPPs are routinely synthesised in vitro by the supersaturation of serum-supplemented cell culture medium with Ca2+ and PO43- ions, as artificially generated CPPs are similar to those isolated from calcified human tissues. Approximate concentration of CPPs in the human blood is 250 particles per µL (2.5 × 108 per L), as measured by fluorescent-labeled bisphosphonate OsteoSense 680EX (IVISense Osteo 680 fluorescent probe). The kinetics of CPP assembling and clearance is far from being fully understood, albeit it evidently depends on the amount of Ca2+ and PO43- ions and acidic serum proteins (especially fetuin-A and albumin), and several reports specified a role of Mg2+ ions, HCO3- ions, and pyrophosphate in these processes as intrinsic Ca2+ antagonists. Although previous studies delineated cytokines overproduced by ECs under CPP treatment (i.e., interleukin-6, interleukin-8, and monocyte chemoattractant protein 1) and itemised other pathological consequences of CPP internalisation (i.e., hampered NO biosynthesis, endothelial-to-mesenchymal transition, and impaired mechanotransduction), a little is known about the proteomic signatures of ECs to calcium stress. Here we for the first time performed an unbiased proteomic profiling of ECs treated with CPPs and revealed relative enrichment of the protein categories related to mitochondrial and lysosomal physiology after the CPP treatment.

Project description:Calciprotein particles (CPPs), assembled as a result of molecular interactions between fetuin-A and nascent calcium phosphate clusters, are indispensable scavengers of excessive Ca2+ and PO43– ions, thus representing an elegant mechanism which regulates mineral homeostasis. Generation of CPPs represents an evolutionary mechanism to prevent blood supersaturation with Ca2+ and PO43– ions (e.g., as a result of bone resorption) and to thwart extraskeletal calcification, a pathological condition which is frequent in patients with chronic kidney disease. It is believed that formation of CPPs accompanied evolution from the appearance of bony fish; the existence of CPPs in more ancient animals remains uncertain. Shortly after emergence, amorphous, spherical, and submicrometer-sized (30-100 nm) primary CPPs (CPP-P) evolve into crystalline, spindle- or needle-shaped, and micrometer-sized (100-300 nm) secondary CPPs (CPP-S). Upon executing their function of aggregating Ca2+ and PO43– ions, CPPs are removed from the circulation by endothelial cells (ECs), monocytes, and liver or spleen macrophages. Internalisation and digestion of CPPs by ECs induce a chain of detrimental events including an increase in cytosolic Ca2+, mitochondrial and endoplasmic reticulum stress, nuclear factor (NF)-κB-mediated transcriptional response and cytokine release, ultimately contributing to the development of pro-inflammatory endothelial activation, chronic low-grade inflammation, and inflammaging. Pathological permeability of dysfunctional endothelium promotes lipid retention and leukocyte extravasation, together contributing to the development of vascular inflammation, proteolytic environment, degradation of basement membrane and internal elastic lamina, and contractile-to-synthetic switch of vascular smooth muscle cells. Such an ensemble of molecular events ultimately induces intimal hyperplasia, vascular remodeling, and atherosclerotic progression. Hence, CPPs act as a double-edged sword which rescues the human body from an acute life-threatening disease (i.e., extraskeletal calcification) at the cost of promoting long-lasting conditions (i.e., endothelial dysfunction and atherosclerosis). Among the several forms of calcium transfer (free Ca2+ ions, colloidal calciprotein monomers, and particulate CPPs), CPPs are the most efficient in delivering calcium stress to ECs because of their deposition and dissolution in lysosomes, followed by a lysosomal membrane permeabilisation and an excessive Ca2+ migration into the cytosol. The molecular pattern of CPP-induced calcium stress within the ECs is well defined and includes elevated expression of cell adhesion molecules (VCAM1, ICAM1, and E-selectin) and endothelial-to-mesenchymal transition transcription factors (SNAI1, SNAI2, TWIST1, and ZEB1) in combination with an endothelial nitric oxide synthase (eNOS) uncoupling. However, our knowledge on extracellular signatures of such endothelial response remains limited to an augmented release of pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, MIF, CXCL1, and MIP-3α/CCL20) and pro- or anti-thrombotic molecules (serpin E1/PAI-1 and uPAR). As EC-secreted bioactive factors (termed angiokines) control vascular tone, maintain hemostasis, and regulate instructive signaling governing local homeostasis within the most organs, decryption and interpretation of endothelial secretome in physiological and pathological conditions is of utmost importance. Discovery of a circulating biomarkers specific for endothelial dysfunction, which can be measured by a routine enzyme-linked immunosorbent assay, might inform on specific disease states and prompt to the respective pharmacological interventions. Here, we aimed at deciphering the secretome of ECs treated with either CPP-P or CPP-S, employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) for the analysis of serum-free cell culture supernatant. For the objective comparison of heterogeneous arterial ECs, we utilised human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) which comprise an innermost lining in atheroprone and atheroresistant blood vessels (coronary artery and internal thoracic artery, respectively).

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Shotgun proteomics analysis of nuclear and mitochondrial from human coronary artery endothelial cells fraction treated with calcioprotein particles

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