Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation
<p>Generation of CPPs in the blood is a distinctive feature of cerebrovascular disease and coronary artery disease. (<b>A</b>) Experimental pipeline assaying human serum propensity to produce CPPs. The assay includes addition of excessive CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> concentrations to the serum followed by 24 h incubation in cell culture conditions and measurement of the optical density at a wavelength of 650 nm (OD<sub>650</sub>), followed by background subtraction (OD<sub>650</sub> of the serum sample without added CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub>). (<b>B</b>) Determination of the optimal amount of added CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> for the assay described in (<b>A</b>). Ascending equimolar concentrations (0.5, 1, 2, 3, 4, and 5 mmol/L) of CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> were added to the serum of healthy individuals (n = 49) followed by OD<sub>650</sub> measurement and background subtraction. The 2 mmol/L of added CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> was a threshold above which all samples showed an OD<sub>650</sub> > 0 after the background subtraction, which indicated CPP formation. (<b>C</b>) Temporal pattern of CPP formation in the setup from (<b>A</b>). Equimolar (2 mmol/L) concentrations of CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> were added to the serum of healthy individuals (n = 42) with the subsequent time-lapse measurement of OD<sub>650</sub> to evaluate CPP formation over time. The average one-half maximal transition time (T<sub>50</sub>) reflects the average time required for reaching one-half of the maximal OD<sub>650</sub> increase across the samples. The one-half maximal OD<sub>650</sub> increase reflects the average OD<sub>650</sub> value equal to half of the maximal OD<sub>650</sub> increase across the samples. (<b>D</b>) Equimolar (2 mmol/L) concentrations of CaCl<sub>2</sub> and Na<sub>2</sub>HPO<sub>4</sub> were added to the serum of healthy individuals (n = 44) and patients with brain ischemia that required carotid endarterectomy (n = 44), ischemic stroke (n = 44), stable angina that required coronary artery bypass graft surgery (n = 44), or myocardial infarction (n = 44), followed by OD<sub>650</sub> measurement and background subtraction. Note an increase in the CPP formation in the brain ischemia, ischemic stroke, and myocardial infarction groups. The tables to the right represent the odds ratios and <span class="html-italic">p</span>-values for the risk of cerebrovascular disease, coronary artery disease, or unstable plaque phenotype in percentiles with the highest and lowest OD<sub>650</sub> increases. (<b>E</b>) Measurement of ionised calcium (Ca<sup>2+</sup>), total protein, and albumin levels in the serum of healthy individuals (n = 88) and patients with cerebrovascular disease (brain ischemia and ischemic stroke combined, n = 88) or coronary artery disease (stable angina and myocardial infarction combined, n = 88). The tables below represent the odds ratios and <span class="html-italic">p</span>-values for the risk of cerebrovascular disease or coronary artery disease in percentiles with the highest and lowest Ca<sup>2+</sup>, total protein, and albumin values. Each dot represents a serum sample collected from one subject. Whiskers indicate ranges, box bounds indicate the 25th–75th percentiles, centre lines indicate the median. <span class="html-italic">p</span>-values are provided above the boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. CPP—calciprotein particles, OD<sub>650</sub>—optical density at 650 nm wavelength.</p> "> Figure 2
<p>Estimated glomerular filtration rate does not define serum CPP generation propensity. Filtration of the subjects in <a href="#ijms-22-12458-f001" class="html-fig">Figure 1</a> by eGFR (selection of those with eGFR ≥ 90 mL/min/m<sup>2</sup> for the subsequent analysis) and repeated statistical analysis of OD<sub>650</sub> increase (CPP generation propensity), serum ionised calcium (Ca<sup>2+</sup>), total protein and albumin concentration, phosphate and fetuin-A level, and age. Note that the results of this repeated statistical analysis do not differ from those without eGFR stratification, i.e., increased CPP generation propensity and serum ionised calcium along with reduced total protein and albumin levels in patients with cerebrovascular disease and coronary artery disease. Note the absence of statistically significant differences in serum phosphate and fetuin-A levels as well as subject age, similar to the general cohorts. Each dot represents a serum sample collected from one subject. Whiskers indicate range, box bounds indicate 25th–75th percentiles, and centre lines indicate median. <span class="html-italic">p</span>-values are provided above boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. CPP—calciprotein particles, eGFR—estimated glomerular filtration rate, and OD<sub>650</sub>—optical density at 650 nm wavelength.</p> "> Figure 3
<p>In vitro synthesised CPP-P are amorphous and round, whereas CPP-S represent crystalline, spindle-shaped nanoparticles with a higher mineral content. (<b>A</b>) Scanning and transmission electron microscopy images showing that CPP-P are spherical particles whereas CPP-S have a spindle- or needle-like shape; (<b>B</b>) 3D reconstruction of CPP-P and CPP-S by atomic force microscopy. (<b>C</b>) Electrophoretic light scattering plot showing that CPP-P and CPP-S have a nearly identical surface charge (from −20 to −25 mV) indicative of their capability to aggregate. (<b>D</b>) Dynamic light scattering measurements demonstrate that CPP-P and CPP-S have a similar particle-size distribution curve, with a diameter range of 100–1000 nm and average diameter of around 500 nm. (<b>E</b>) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis with subsequent silver staining show that CPP-P and CPP-S have a similar protein profile. (<b>F</b>) Atomic emission spectroscopy confirms that both CPP-P and CPP-S contain calcium and phosphorus. (<b>G</b>) Energy-dispersive X-ray spectroscopy shows higher carbon content in CPP-P but an increased proportion of calcium and phosphorus in CPP-S. (<b>H</b>) CHNSO analysis allows detection of nitrogen, carbon, and hydrogen in both CPP-P and CPP-S, also documenting higher amounts of carbon in CPP-P than in CPP-S. (<b>I</b>) Fourier transform infrared spectroscopy identifies phosphate (PO<sub>4</sub><sup>3−</sup>), carbonate (CO<sub>3</sub><sup>2−</sup>), and hydroxyl (OH<sup>−</sup>) groups in both CPP-P and CPP-S. Note the higher phosphate content in CPP-S compared with CPP-P. (<b>J</b>) X-ray powder diffraction analysis demonstrates that both CPP-P and CPP-S consist of hydroxyapatite (Ca<sub>10</sub>(PO<sub>4</sub>)<sub>6</sub>(OH)<sub>2</sub> and carbonate-hydroxyapatite (Ca<sub>10</sub>(PO<sub>4</sub>)<sub>3</sub>(CO<sub>3</sub>)<sub>3</sub>(OH)<sub>2</sub>). (<b>K</b>) Selected area diffraction patterns indicate the lower crystallinity of CPP-P in comparison with CPP-S. CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, and EDX—energy-dispersive X-ray spectroscopy.</p> "> Figure 4
<p>CPPs induce intimal hyperplasia in both injured and intact rat aortas. (<b>A</b>) H&E-stained aortas from balloon-injured rats treated by a single intravenous injection of NaCl, MPPs, CPP-P, or CPP-S (900 μL of particles, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 450 μg calcium or 1.08 × 10<sup>6</sup> OsteoSense 680EX-positive PKH67-negative events) immediately postoperation (n = 10 rats per group, 40 rats in total). Note the intimal hyperplasia in rats treated with CPP-P and CPP-S. (<b>B</b>) Proportion of rats with intimal hyperplasia and neointimal area calculated upon the analysis of H&E-stained aortas. Each dot represents an aortic section from one rat. Note that the zero neointimal area corresponds to the absence of neointima. The dotted line indicates a threshold value for the definition of intimal hyperplasia (≥0.5 × 10<sup>4</sup> μm<sup>2</sup>). Whiskers indicate range, box bounds indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values are provided above boxes (statistically significant differences are marked by asterisks), Pearson’s chi-squared test with Yates’s correction for continuity or Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (<b>C</b>) H&E-stained aortas from rats intravenously injected with NaCl, MPPs, CPP-P, or CPP-S (900 μL of particles per injection, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 450 μg calcium or 1.08 × 10<sup>6</sup> OsteoSense 680EX-positive PKH67-negative events) once or thrice a week (n = 10 rats per group, 70 rats in total) for a total duration of 5 weeks. Note the intimal hyperplasia in rats treated with CPP-P and CPP-S. (<b>D</b>) Semi-quantitative image analysis of H&E-stained aortas of rats from the experiment in (<b>C</b>). Shown is the proportion of rats with intimal hyperplasia. Pearson’s chi-squared test with Yates’s correction for continuity. (<b>E</b>) Association of CPP-induced intimal hyperplasia with flow patterns characteristic of different aortic segments (aortic arch with a turbulent flow and descending aorta characterised by a laminar flow). Shown is the proportion of rats with intimal hyperplasia. Pie charts represent the proportions of laminar and turbulent segments (i.e., descending aorta and aortic arch, respectively) among those with intimal hyperplasia upon CPP-P or CPP-S administration. Pearson’s chi-squared test with Yates’s correction for continuity. H&E—haematoxylin and eosin, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, and OD<sub>650</sub>—optical density at 650 nm wavelength.</p> "> Figure 5
<p>CPPs induce adventitial and perivascular inflammation which correlates with intimal hyperplasia. (<b>A</b>) Representative images of adventitial and perivascular vasa vasorum (VV) in the aortas of rats that underwent balloon angioplasty followed by daily intravenous injections of NaCl, MPPs, CPP-P, or CPP-S (900 μL of particles per injection, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 450 μg calcium or 1.08 × 10<sup>6</sup> OsteoSense 680EX-positive PKH67-negative events) for 5 days (n = 5 rats per group, 20 rats in total). Rats were euthanised 5 weeks postoperation with subsequent backscattered scanning electron microscopy examination of the injured (n = 7–12) and intact (n = 4–5) aortic segments. Red circles denote areas with VV. (<b>B</b>) Semi-quantitative image analysis of the VV from the experiment in (<b>A</b>). VV density was calculated for both VV number and VV area (area of the adventitial and perivascular adipose tissue area per one vessel (μm<sup>2</sup>) or 1 μm<sup>2</sup> of the VV area, respectively). (<b>C</b>) Representative images of adventitial and perivascular macrophage clusters (MCs) in the rat aortas from the experiment in (<b>A</b>). Red circles denote MCs. (<b>D</b>) Semi-quantitative image analysis of the MCs from the experiment in (<b>C</b>). MC density was calculated in similar to VV density. (<b>E</b>) H&E-stained aortas containing MCs from balloon-injured rats treated by a single intravenous injection of NaCl, MPP, CPP-P, or CPP-S immediately postoperation (n = 10 rats per group, 40 rats in total). Red quadrants within the whole section images (left) indicate regions of interest with MCs (right). Insets confirm positive staining of MCs for a macrophage marker F4/80 and myeloperoxidase (MPO), an enzyme reflecting macrophage activity. MCs are also negative for T cell marker CD3. (<b>F</b>) Semi-quantitative image analysis of the MCs in the rat aortas from the experiment in €. Each dot represents an aortic section from one rat. Whiskers indicate the range, box bounds indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values are provided above boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli or Mann–Whitney U-test. (<b>G</b>) Primary human perivascular adipocytes were treated with PBS or CPP-P (50 μL of particles per well on a 24-well plate, OD<sub>650</sub> = 0.08–0.10, equal to 25 μg calcium or 0.6 × 10<sup>5</sup> OsteoSense 680EX-positive PKH67-negative events) for 24 h. Conditioned media and cell lysate were profiled for adipokines and cytokines. Numbers indicate densitometry relative to PBS group. Red and green circles indicate increase and decrease in the adipokine/cytokine level, respectively. Note a decrease in adiponectin in CPP-P-treated cells. VV—vasa vasorum, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, MCs—macrophage clusters, H&E—haematoxylin and eosin, MPO—myeloperoxidase, and PBS—phosphate-buffered saline.</p> "> Figure 6
<p>CPP-P and CPP-S inflict cell death in culture. (<b>A</b>) Phase contrast microscopy images of primary human coronary artery endothelial cells (HCAECs) and human internal thoracic artery endothelial cells (HITAECs) treated with PBS, MPPs, CPP-P, or CPP-S (100 μL of particles per well of a 6-well plate, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 50 μg calcium or 1.2 × 10<sup>5</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h. (<b>B</b>) Indicated cells stained for Hoechst 33342 (blue) and EtBr (red). EtBr-positive staining indicates dead cells. (<b>C</b>) Quantification of viable cells by colorimetric proliferation and viability assay after treatment with PBS, MPPs, CPP-P, or CPP-S (10 μL of particles per well of a 96-well plate, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 5 μg calcium or 1.2 × 10<sup>4</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h (n = 24 wells per group). The proportions of viable cells were calculated in relation to the PBS group. (<b>D</b>) Quantification of dead cells from the experiment in B (n = 11 wells per group). (<b>C</b>,<b>D</b>) Each dot represents one well of the culture plate. Whiskers indicate the range, box bounds indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values are provided above boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. HCAECs—human coronary artery endothelial cells, HITAECs—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, OD<sub>650</sub>—optical density at 650 nm wavelength, and EtBr—ethidium bromide.</p> "> Figure 7
<p>Lysosome-dependent cell death defines specific toxicity of CPPs for endothelial cells. (<b>A</b>) Transmission electron microscopy images of HCAECs treated with PBS or CPPs for 4 h. Inserts show internalised CPPs. (<b>B</b>) Backscattered scanning electron microscopy of HCAECs treated with PBS, MPPs, CPP-P, or CPP-S for 1 h. Note the electron-dense dots indicative of MPP, CPP-P, and CPP-S internalisation. Energy-dispersive X-ray spectroscopy detected Ca content when applied to these electron-dense dots in the cells treated with CPP-P or CPP-S. (<b>C</b>) Fluorescent microscopy of HCAECs treated with FITC-labelled CPPs (green) for 1 or 4 h and stained with Hoechst 33342 (nuclear stain, blue) and LysoTracker Red (lysosomes, red). Note the colocalisation of FITC-labelled CPPs with lysosomes 1 h post-treatment and partial translocation of FITC-labelled CPPs into the cytosol 4 h post-treatment. (<b>D</b>) HCAECs were treated with vehicle or ascending concentrations of bafilomycin A1 in a time-course manner and incubated in the presence of MPPs or CPPs (10 μL of particles per well of a 96-well plate, OD<sub>650</sub> = 0.08–0.10, for CPPs: equal to 5 μg calcium or 1.2 × 10<sup>4</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 or 24 h (n = 12 wells per group) with subsequent quantitation of viable cells. Bottom-right graph demonstrates the ratio of viable cells after exposure to CPPs or MPPs. Each dot represents one well of the culture plate. Whiskers indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values provided above the graphs (statistically significant differences are marked by asterisks), Mann–Whitney U-test. (<b>E</b>) X-ray powder diffraction analysis demonstrates that MPPs and CPPs consist of highly soluble magnesium phosphate hydrate and less soluble hydroxyapatite, respectively. (<b>F</b>) HCAECs were treated with PBS or CPPs for 1 h and stained with Hoechst 33342 (nuclear stain, blue), fluo-3 AM (Ca<sup>2+</sup> indicator, green), and LysoTracker Red (lysosomes, red). Note the colocalisation of Ca<sup>2+</sup> with lysosomes and their partial translocation into the cytosol. (<b>G</b>) HCAECs were cultured in the presence of PBS, MPPs, CPP-P, or CPP-S (100 μL of particles per well of a 6-well plate, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 50 μg calcium or 1.2 × 10<sup>5</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h and immunoblotted for the indicated proteins. Histone H3 was used as the loading control. Numbers indicate densitometry relative to the PBS group. HCAECs—human coronary artery endothelial cells, PBS—phosphate-buffered saline, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, FITC—fluorescein isothiocyanate, OD<sub>650</sub>—optical density at 650 nm wavelength, Veh—vehicle control, Baf A1—bafilomycin A1, PARP-1—poly [ADP-ribose] polymerase 1, and PAI-1—plasminogen activator inhibitor 1.</p> "> Figure 8
<p>CPPs induce endothelial activation and endothelial-to-mesenchymal transition. (<b>A</b>) HCAECs and HITAECs were cultured in the presence of PBS, MPPs, CPP-P, or CPP-S (100 μL of particles per well of a 6-well plate, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 50 μg calcium or 1.2 × 10<sup>5</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h, and total RNA was extracted with the following expression profiling for the indicated genes (n = 3 wells per group). Heat map shows differentially expressed genes (fold change > 2) between groups. (<b>B</b>) HCAECs and HITAECs were cultured in the presence of PBS, MPPs, CPP-P, or CPP-S (100 μL of particles per well of a 6-well plate, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 50 μg calcium or 1.2 × 10<sup>5</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h. Conditioned media were profiled for interleukin-6 and -8 using an enzyme-linked immunosorbent assay (n = 6 wells per group). Each dot represents one well of the culture plate. Whiskers indicate the range, box bounds indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values are provided above the boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (<b>C</b>) Conditioned media and cell lysate from HCAECs and HITAECs from the experiment in (<b>B</b>) were profiled using a cytokine array. Note the increase in interleukin-8 in both the supernatant and cell lysate as well as the higher contents of macrophage migration inhibitory factor (MIF) and chemokine (C-X-C motif) ligand 1 (CXCL1) in the supernatant. Numbers indicate densitometry relative to the PBS group. (<b>D</b>) HCAECs and HITAECs were cultured in the flow system for 24 h with subsequent treatment with PBS, MPPs, CPP-P, or CPP-S (500 μL of particles per flow system unit, OD<sub>650</sub> = 0.08–0.10, for CPP-P and CPP-S: equal to 250 μg calcium or 0.6 × 10<sup>6</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h and incubation with CellTracker Green CMFDA labelled peripheral blood mononuclear cells (PBMCs) for 1 h. Representative images. Note the considerable adhesion of PBMCs to CPP-P and CPP-S-treated cells. (<b>E</b>) Semi-quantitative image analysis of the experiment in (<b>D</b>). Each dot represents one image (n = 40 images per group). Whiskers indicate the 25th–75th percentiles, and centre lines indicate the median. <span class="html-italic">p</span>-values are provided above boxes (statistically significant differences are marked by asterisks), Kruskal–Wallis test with post hoc false discovery rate correction by the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. (<b>F</b>) HCAECs and HITAECs were treated with PBS or CPP-P (1000 μL of particles per T-75 flask, OD<sub>650</sub> = 0.08–0.10, equal to 500 μg calcium or 1.2 × 10<sup>6</sup> OsteoSense 680EX-positive PKH67-negative events) for 4 h with further immunoblotting for proteins reflective of endothelial activation and endothelial-to-mesenchymal transition. Note increases in VCAM1 and ICAM1, cell adhesion molecules mediating leukocyte attachment, and Snail and Slug, the key transcription factors regulating endothelial-to-mesenchymal transition. β-tubulin was used as the loading control. Numbers indicate densitometry relative to the PBS group. HCAECs—human coronary artery endothelial cells, HITAECs—human internal thoracic artery endothelial cells, PBS—phosphate-buffered saline, MPPs—magnesiprotein particles, CPP-P—primary calciprotein particles, CPP-S—secondary calciprotein particles, OD<sub>650</sub>—optical density at 650 nm wavelength, CMFDA—5-chloromethylfluorescein diacetate, VCAM1—vascular cell adhesion molecule 1, ICAM1—intercellular cell adhesion molecule 1, KDR—kinase insert domain receptor, VE-cadherin—vascular endothelial cadherin, and N-cadherin—neural cadherin.</p> "> Figure 9
<p>An illustration depicting the putative mechanism of CPP-induced vascular injury in patients with cardiovascular disease. (<b>A</b>) Physiological scenario in healthy individuals; (<b>B</b>) pathological scenario in patients with cerebrovascular disease or coronary artery disease and reduced Ca<sup>2+</sup>-binding capacity. Supersaturation with Ca<sup>2+</sup> ions due to reduced serum Ca<sup>2+</sup>-binding capacity leading to CPP aggregation in the blood. CPPs are internalised by the endothelial cells causing their lysosome-dependent death, endothelial activation, and endothelial-to-mesenchymal transition. This provokes leukocyte adhesion and the excessive release of proinflammatory cytokines interleukin-6 and -8, collectively promoting chronic inflammation and neointima formation. Furthermore, CPPs cause adventitial and perivascular inflammation reflected by the expansion of the vasa vasorum and macrophage clusters, which also contributes to intimal hyperplasia and disrupted vascular homeostasis.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Atherosclerotic Vascular Disease Is Characterised by Augmented CPP Formation as a Consequence of Disturbed Mineral Homeostasis
2.2. Recapitulation of the Physiological Scenario by Synthesising Primary and Secondary CPPs
2.3. CPPs Provoke Intimal Hyperplasia and Vascular Inflammation in the Absence of Other Cardiovascular Risk Factors
2.4. Lysosome-Dependent Cell Death Defines the Toxicity of CPPs toward Endothelial Cells
2.5. CPPs Cause Endothelial Activation and Endothelial-to-Mesenchymal Transition
3. Discussion
- We did not apply the T50 test, which reflects the rate of transformation of CPP-P to CPP-S in the human serum, as we think the total propensity of serum to form CPPs (measured by OD650 increment) is a suitable and pathophysiologically relevant alternative that might be less sophisticated for routine clinical use. The T50 test has its advantages (e.g., validation of multiple patient cohorts), yet the mineral stress test (OD650 increase) applied in this study is not contradictory and represents another variation in serum calcification propensity measurement.
- The concentration of +2 mmol/L CaCl2 and Na2HPO4 added is supraphysiological and such excessive Ca/P concentrations are rarely encountered, except in patients with severe osteopenia/osteoporosis and hyperparathyroidism who frequently show hypercalcemia and those with ESRD who generally suffer from hyperphosphatemia. Yet, this Ca/P increase represents a mineral stress test for the measurement of serum’s propensity to form CPPs; lower amounts of added CaCl2 and Na2HPO4 concentrations inconsistently triggered CPP generation. Increased CPP generation propensity (higher OD650 increase values) was associated with higher ionised calcium (Ca2+) and lower albumin, thereby reflecting disturbed mineral homeostasis in cohorts of patients with coronary artery disease and cerebrovascular disease compared with healthy blood donors.
- In our study, we did not perform animal section immunostainings as these sections were formalin-fixed for significantly longer than 24 h and therefore were unsuitable for IHC-P or IHC-Fr applications, although electron microscopy analysis well-distinguishes blood microvessels from the background by the combination of clearly visible vessel lumen, endothelial layer, and (optionally) red blood cells within the lumen. In addition, macrophages also have morphological patterns distinct from other adventitial cell populations (e.g., fibroblasts and lymphocytes), in particular when assembled into clusters, which is not characteristic for fibroblasts. IHC stainings of the aortic sections from another experiment showed positive staining of MCs for F4/80 and myeloperoxidase but not for T lymphocyte CD3 marker (Figure 5E, insets).
- We did not study CPP’s effects on vascular smooth muscle cells. However, we stained aortas from both animal models with alizarin red S and found no signs of vascular calcification, suggesting that the osteogenic differentiation of vascular smooth muscle cells is not among the major consequences of CPP administration in vivo. In a balloon injury model, endothelial denudation and mechanical vascular injury collectively led to intimal hyperplasia because of damaged internal elastic lamina and the possible proliferative activation of vascular smooth muscle cells. We suggest that exposure of balloon-injured blood vessel to CPPs might also contribute to the contractile-to-synthetic phenotypic switch of vascular smooth muscle cells, although we could not confirm this because the sections could not be used for immunohistochemistry. Whether this scenario occurs in intact blood vessels is currently unclear, whereas endothelial dysfunction might result in pathological paracrine signalling to adjacent vascular smooth muscle cells.
4. Materials and Methods
4.1. Evaluation of Serum Propensity for Calciprotein Particle (CPP) Formation
4.2. Artificial Synthesis and Quantification of CPPs
4.3. Electron and Atomic Force Microscopy
4.4. Measurement of Particle-Size Distribution and Surface Charge
4.5. Chemical Profiling
4.6. Animal Models
4.7. Cell Culture
4.8. Cytotoxicity Assays
4.9. Internalisation Assays
4.10. Lysosome Permeabilisation Assay
4.11. Ca2+ Translocation Assay
4.12. Assessment of Oxidative Stress
4.13. Thiobarbituric Acid Reactive Substances (TBARS) Assay
4.14. Gene Expression Profiling
4.15. Cytokine Secretion Profiling
4.16. Leukocyte Adhesion Assay
4.17. Western Blotting
4.18. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Shishkova, D.K.; Velikanova, E.A.; Bogdanov, L.A.; Sinitsky, M.Y.; Kostyunin, A.E.; Tsepokina, A.V.; Gruzdeva, O.V.; Mironov, A.V.; Mukhamadiyarov, R.A.; Glushkova, T.V.; et al. Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation. Int. J. Mol. Sci. 2021, 22, 12458. https://doi.org/10.3390/ijms222212458
Shishkova DK, Velikanova EA, Bogdanov LA, Sinitsky MY, Kostyunin AE, Tsepokina AV, Gruzdeva OV, Mironov AV, Mukhamadiyarov RA, Glushkova TV, et al. Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation. International Journal of Molecular Sciences. 2021; 22(22):12458. https://doi.org/10.3390/ijms222212458
Chicago/Turabian StyleShishkova, Daria K., Elena A. Velikanova, Leo A. Bogdanov, Maxim Yu. Sinitsky, Alexander E. Kostyunin, Anna V. Tsepokina, Olga V. Gruzdeva, Andrey V. Mironov, Rinat A. Mukhamadiyarov, Tatiana V. Glushkova, and et al. 2021. "Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation" International Journal of Molecular Sciences 22, no. 22: 12458. https://doi.org/10.3390/ijms222212458