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Proteomics of endothelial cell secretome after exposure to calciprotein particles

ABSTRACT: 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).

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Internal Thoracic Artery, Endothelial Cell Of Coronary Artery

DISEASE(S): Calcinosis,Cardiovascular System Disease,Atherosclerosis

SUBMITTER: Egor Repkin

LAB HEAD: Anton Kutikhin

PROVIDER: PXD055909 | Pride | 2024-10-24

REPOSITORIES: Pride

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			Action	DRS
	131_Slot1-16_1_5281.d.zip	Other
	131_Slot1-16_1_5281.pepXML	Pepxml
	131_Slot1-16_1_5281_uncalibrated.mzML	Mzml
	132_Slot1-7_1_5272.d.zip	Other
	132_Slot1-7_1_5272.pepXML	Pepxml

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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:Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dys-functional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the mor-phine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothe-lial cells as well as on their associated EVs. Isolation of EVs was carried out using an affini-ty-based method. Several orthogonal techniques (NTA, western blotting and proteomics analy-sis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modu-lations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 pro-teins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the “cell adhesion and extracellular matrix remodeling” process and the “HIF1 pathway”, a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings high-lighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient cir-culation using these “signature pathways”.

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Proteomics of endothelial cell secretome after exposure to calciprotein particles

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