TOXICITY OF SPHERICAL AND NEEDLE-SHAPED CALCIUM PHOSPHATE BIONS TO INJURED INTIMA OF THE RAT ABDOMINAL AORTA

Цель исследования - оценка токсического действия сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на предварительно поврежденную интиму аорты крыс. Методика. Токсическое действие сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на поврежденную интиму брюшной аорты крыс линии Wistar (n = 10 на группу) оценивали путем их однократного внутривенного введения после баллонной ангиопластики с эксплантацией поврежденного участка аорты через 5 нед. Биоптаты анализировали: 1) классическими гистологическими методами (окрашивание гематоксилин-эозином, ализариновым красным, по Вейгерту-ван Гизону и по Расселлу-Мовату); 2) иммунофлюоресцентным окрашиванием криосрезов (сочетанное окрашивание на CD31 и CD34, на CD31 и α-гладкомышечный актин (α-ГМА), на виментин и α-ГМА, на коллаген IV типа и α-ГМА). Для оценки влияния системного воспаления на КФБ-индуцированную эндотелиотоксичность определяли содержание моноцитарного хемоаттрактантного белка (МСР-1/CCL2) и церулоплазмина в сыворотке крови прооперированных крыс посредством иммуноферментного анализа. Результаты. Сферические кальций-фосфатные бионы и игольчатые кальций-фосфатные бионы вызывали выраженную гипертрофию интимы брюшной аорты в 90% (9 из 10 крыс) и 80% случаев (8 из 10 крыс) соответственно, в то время как частота гипертрофии в группе контрольных крыс составила лишь 10% (1 из 10 крыс). Неоинтима при экспозиции интимы брюшной аорты обоим типам бионов характеризовалась переходом фенотипа клеток мезенхимального ряда с контрактильного (α-ГМА-положительные и виментин-отрицательные гладкомышечные клетки) и неактивного (α-ГМА-отрицательные и виментин-положительные фибробласты) на активный синтетический (α-ГМА- и виментин-положительные клетки), что приводило к формированию значительных количеств экстрацеллюлярного матрикса. Внутривенное введение сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов не приводило к изменению уровней МСР-1/CCL2 и церулоплазмина в сыворотке крови, что свидетельствовало об отсутствии их возможного влияния на развитие системного воспалительного ответа. Заключение. Внутривенное введение кальций-фосфатных бионов после повреждения интимы брюшной аорты крыс путем баллонной ангиопластики вызывает развитие гипертрофии интимы, частота и выраженность которой не зависит от формы кальций-фосфатных бионов и которая характеризуется переходом фенотипа клеток мезенхимального ряда из контрактильного/неактивного на активный синтетический. При этом эндотелиотоксическое действие кальций-фосфатных бионов обусловлено их непосредственным воздействием на эндотелий, а не развитием системного воспаления. Purpose. To compare toxicity of spherical calcium phosphate bions (SCPB) and needle-shaped calcium phosphate bions (NCPB) to injured intima of rat aortas. Methods. Toxicity of SCPB and NCPB to injured abdominal aortas of Wistar rats (n = 10 per group) was evaluated using intravenous administration of the bions after balloon angioplasty. Rats were sacrificed five weeks postoperation, and an injured aortic segment was excised. Tissue preparations were stained with hematoxylin and eosin, alizarin red S, Weigert-van Gieson, and Movat’s pentachrome stains. Selected tissue samples were then examined using combined immunofluorescence staining (CD31/CD34, CD31/α-smooth muscle actin (α-SMA), α-SMA/vimentin, and α-SMA/collagen IV). Possible influence of systemic inflammation on CPB-induced endothelial toxicity was assessed by measuring monocyte chemoattractant protein-1 and ceruloplasmin in rat serum using the enzyme-linked immunosorbent assay. Results. Intravenous administration of SCPB or NCPB provoked intimal hyperplasia in 90% (9 of 10) and 80% (8 of 10) of rats vs. 10% (1 of 10) in the control group. The neointima was characterized by a phenotypic switch of mesenchymal cells, i.e. transition of a contractile (α-SMA-positive, vimentin-negative vascular smooth muscle cells) and quiescent (α-SMA-negative vimentin-positive fibroblasts) to an active synthetic phenotype (double-positive cells), which resulted in deposition of the extracellular matrix. Neither SCPB nor NCPB changed serum levels of pro-inflammatory molecules, МСР-1/CCL2, and ceruloplasmin. Conclusions. Intravenous administration of CPB upon balloon-induced vascular injury caused intimal hyperplasia regardless of the CPB shape. Hyperplasia foci were characterized by a switch of mesenchymal cells from a contractile/quiescent to an active synthetic phenotype. Endothelial toxicity of CPBs was defined by their direct cytotoxic action rather than induction of systemic inflammation.

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Calcium-phosphate bions do specifically induce hypertrophy of damaged intima in rats

Russian Journal of Cardiology ◽

10.15829/1560-4071-2018-9-33-38 ◽

2018 ◽

pp. 33-38 ◽

Cited By ~ 1

Author(s):

D. K. Shishkova ◽

E. A. Velikanova ◽

E. O. Krivkina ◽

A. V. Mironov ◽

Yu. A. Kudryavtseva ◽

...

Keyword(s):

Smooth Muscle ◽

Calcium Phosphate ◽

Smooth Muscle Cells ◽

Abdominal Aorta ◽

Muscle Cells ◽

Mesenchymal Cells ◽

Enzyme Analysis ◽

Collagen Type Iv ◽

Alizarin Red

Aim. To evaluate specificity of endothelial toxicity of calcium-phosphate bions (CPB) in vivo.Material and methods. Toxicity of calcium-phosphate bions and magnesiumphosphate bions (MPB) in relation to intima of abdominal aorta of the Wistar rats, was assessed by single intravenous injection after balloon angioplastics with further explanting of aortas in five weeks. Bioptates were analyzed: 1) with classical histological methods (hematoxilin-eosine, alizarin red, van Gison, Russell-Movat) with light microscopy; 2) immune fluorescence coloring of cryoslices (combinational coloring for marker of mature endothelial cells CD31 and marker of progenitory CD34, for CD31 and marker of vascular smooth muscle cells α-smoothmuscle actin (α-SMA), for vimentin and α-SMA, for extracellular matrix marker collagen type IV and α-SMA, after all colorings there was additional nuclear 4’,6-diamidine-2-phenylindol color) with further confocal microscopy. In all animals the blood was collected with serum extraction for systemic inflammation molecules analysis, as chemoattractant protein (МСР-1/CCL2) and ceruloplasmin via the immune enzyme analysis.Results. With the difference from CPB, MPB did not lead to intimal hypertrophy in abdominal aorta in rats. Shaping of neointima in aorta is related with CPB-induced endothelium damage that induces a phenotype shift in mesenchymal cells (smooth muscle cells and fibroblasts) from contractile (for smooth muscle) and non-active (for fibroblasts) towards synthetizing.Conclusion. Intravenous load of MPB did not lead to intimal hypertrophy that witness on specificity of endothelial toxicity of CPB, with phenotypical shift of the mesenchymal cells in neointima.

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The mTOR/p70 S6K1 pathway regulates vascular smooth muscle cell differentiation

AJP Cell Physiology ◽

10.1152/ajpcell.00201.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. C507-C517 ◽

Cited By ~ 157

Author(s):

Kathleen A. Martin ◽

Eva M. Rzucidlo ◽

Bethany L. Merenick ◽

Diane C. Fingar ◽

David J. Brown ◽

...

Keyword(s):

Protein Synthesis ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Intimal Hyperplasia ◽

Transcriptional Control ◽

Matrix Protein ◽

Kinase Inhibitors ◽

Extracellular Matrix Protein ◽

Induced Expression ◽

Synthetic Phenotype

Vascular smooth muscle cells (VSMC) in mature, normal blood vessels exhibit a differentiated, quiescent, contractile morphology, but injury induces a phenotypic modulation toward a proliferative, dedifferentiated, migratory phenotype with upregulated extracellular matrix protein synthesis (synthetic phenotype), which contributes to intimal hyperplasia. The mTOR (the mammalian target of rapamycin) pathway inhibitor rapamycin inhibits intimal hyperplasia in animal models and in human clinical trials. We report that rapamycin treatment induces differentiation in cultured synthetic phenotype VSMC from multiple species. VSMC treated with rapamycin assumed a contractile morphology, quantitatively reflected by a 67% decrease in cell area. Total protein and collagen synthesis were also inhibited by rapamycin. Rapamycin induced expression of the VSMC differentiation marker contractile proteins smooth muscle (SM) α-actin, calponin, and SM myosin heavy chain (SM-MHC), as observed by immunoblotting and immunohistochemistry. Notably, we detected a striking rapamycin induction of calponin and SM-MHC mRNA, suggesting a role for mTOR in transcriptional control of VSMC gene expression. Rapamycin also induced expression of the cyclin-dependent kinase inhibitors p21cip and p27kip, consistent with cell cycle withdrawal. Rapamycin inhibits mTOR, a signaling protein that regulates protein synthesis effectors, including p70 S6K1. Overexpression of p70 S6K1 inhibited rapamycin-induced contractile protein and p21cip expression, suggesting that this kinase opposes VSMC differentiation. In conclusion, we report that regulation of VSMC differentiation is a novel function of the rapamycin-sensitive mTOR signaling pathway.

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Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/β-catenin pathway

Journal of Molecular Histology ◽

10.1007/s10735-021-10012-2 ◽

2021 ◽

Author(s):

Yun Zhou ◽

Li-Long Wei ◽

Rui-Ping Zhang ◽

Cheng-Wu Han ◽

Yongtong Cao

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Osteoblastic Differentiation ◽

Alizarin Red S ◽

Regulation Mechanism ◽

Alizarin Red ◽

Globular Adiponectin

AbstractLipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that β-glycerophosphate (β-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in β-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/β-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/β-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/β-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

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Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis

TH Open ◽

10.1055/s-0041-1725042 ◽

2021 ◽

Vol 05 (02) ◽

pp. e113-e124

Author(s):

Elsa Wilma Böhm ◽

Maria Pavlaki ◽

Georgios Chalikias ◽

Dimitrios Mikroulis ◽

George S. Georgiadis ◽

...

Keyword(s):

Calcium Phosphate ◽

Aortic Valve ◽

Carotid Artery ◽

Vascular Tissue ◽

Osteoblastic Differentiation ◽

Aortic Aneurysms ◽

Vascular Lesions ◽

Erythrocyte Membranes ◽

Intraplaque Hemorrhage ◽

Alizarin Red

Abstract Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied. Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis (n = 46), atherosclerotic carotid artery plaques (n = 9), and abdominal aortic aneurysms (n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson–Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals. Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.

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Smooth muscle cell-specific knockout of interferon gamma (IFN-γ) receptor attenuates intimal hyperplasia via STAT1-KLF4 activation

Life Sciences ◽

10.1016/j.lfs.2021.119651 ◽

2021 ◽

pp. 119651

Author(s):

Lu He ◽

Kun Gao ◽

Hongxia Liu ◽

Jing Wang ◽

Xinwei Li ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cell ◽

Interferon Gamma ◽

Muscle Cell ◽

Intimal Hyperplasia ◽

Ifn Γ

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RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

Calcified Tissue International ◽

10.1007/s00223-021-00833-4 ◽

2021 ◽

Author(s):

Jingjing Cong ◽

Bei Cheng ◽

Jinyu Liu ◽

Ping He

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Glycogen Synthase ◽

Critical Role ◽

Transcriptional Enhancer ◽

Alizarin Red ◽

Gsk 3Β ◽

Enhancer Factor

AbstractVascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

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Enhanced Osteogenesis of Dental Pulp Stem Cells In Vitro Induced by Chitosan–PEG-Incorporated Calcium Phosphate Cement

Polymers ◽

10.3390/polym13142252 ◽

2021 ◽

Vol 13 (14) ◽

pp. 2252

Author(s):

Jae Eun Kim ◽

Sangbae Park ◽

Woong-Sup Lee ◽

Jinsub Han ◽

Jae Woon Lim ◽

...

Keyword(s):

Stem Cells ◽

Calcium Phosphate ◽

Zeta Potential ◽

Mechanical Strength ◽

Calcium Phosphate Cement ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Ion Trapping ◽

Alizarin Red

The use of bone graft materials is required for the treatment of bone defects damaged beyond the critical defect; therefore, injectable calcium phosphate cement (CPC) is actively used after surgery. The application of various polymers to improve injectability, mechanical strength, and biological function of injection-type CPC is encouraged. We previously developed a chitosan–PEG conjugate (CS/PEG) by a sulfur (VI) fluoride exchange reaction, and the resulting chitosan derivative showed high solubility at a neutral pH. We have demonstrated the CPC incorporated with a poly (ethylene glycol) (PEG)-grafted chitosan (CS/PEG) and developed CS/PEG CPC. The characterization of CS/PEG CPC was conducted using Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). The initial properties of CS/PEG CPCs, such as the pH, porosity, mechanical strength, zeta potential, and in vitro biocompatibility using the WST-1 assay, were also investigated. Moreover, osteocompatibility of CS/PEG CPCs was carried out via Alizarin Red S staining, immunocytochemistry, and Western blot analysis. CS/PEG CPC has enhanced mechanical strength compared to CPC, and the cohesion test also demonstrated in vivo stability. Furthermore, we determined whether CS/PEG CPC is a suitable candidate for promoting the osteogenic ability of Dental Pulp Stem Cells (DPSC). The elution of CS/PEG CPC entraps more calcium ion than CPC, as confirmed through the zeta potential test. Accordingly, the ion trapping effect of CS/PEG is considered to have played a role in promoting osteogenic differentiation of DPSCs. The results strongly suggested that CS/PEG could be used as suitable additives for improving osteogenic induction of bone substitute materials.

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Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway

Blood ◽

10.1182/blood.v82.1.66.bloodjournal82166 ◽

1993 ◽

Vol 82 (1) ◽

pp. 66-76 ◽

Cited By ~ 261

Author(s):

MC Galmiche ◽

VE Koteliansky ◽

J Briere ◽

P Herve ◽

P Charbord

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Stromal Cells ◽

Muscle Cells ◽

Mesenchymal Cells ◽

Myoid Cells ◽

Marrow Cultures

In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle- specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha- SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.

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