Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials

<div><p>Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1⁺ (Sca-1⁺/PDGFRα⁺ and Sca-1⁺/PDGFRα⁻) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1⁻ (Sca-1⁻/PDGFRα⁺ and Sca-1⁻/PDGFRα⁻) progenitor cells. Among Sca-1⁺ progenitor populations, Sca-1⁺/PDGFRα⁻ cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1⁺/PDGFRα⁺ cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1⁺/PDGFRα⁻ cells preferentially differentiated into osteoclast-like cells. Sca-1⁺ progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1⁺ calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1⁺ progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1⁺/PDGFRα⁻ cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1⁺/PDGFRα⁻ cells into Apoe^−/− mice increased the severity of calcified atherosclerotic plaques. However, Sca-1⁺/PDGFRα⁻ cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1⁺/PDGFRα⁻ cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.</p> </div

Calcifying progenitor cells induce calcification in atherosclerotic plaques of Apoe^−/− mice.

<p>(A) Calcium accumulation levels in mouse arteries according to diet. *<i>P</i><0.005 versus C57 fed a normal diet (<i>n</i> = 10 per group). (B) Experimental timeline. (C) Calcium accumulation levels in the aortas of Apoe^−/− mice according to BM cell type injected and PPARγ activation. *<i>P</i><0.001 versus PBS-treated group. ‡<i>P</i><0.001 versus mice injected with BM Sca-1⁺/PDGFRα⁻ cells. (D) Atherosclerotic plaque with MT staining. Bars: 200 µm. (E) Calcification induction in atherosclerotic plaques was detected by von Kossa staining. Arrows indicate calcified areas. Bars: 200 µm. (F) Atherosclerotic calcification plaque immunostained with osteocalcin, an osteoblastic marker, and RANK, an osteoclastic marker, to determine the fates of infiltrating BM-derived calcifying progenitor cells in the presence/absence of PPARγ activation. Bars: 20 µm. (G) Image enlargement showing osteocalcin and RANK immunostaining. Bars: 20 µm. (H) Osteoblastic, osteoclastic, and bidirectional cell counts in the artery. GFP⁺Sca-1⁺/PDGFRα⁺ cells primarily expressed osteoblast markers. GFP⁺Sca-1⁺/PDGFRα⁻ cells mainly differentiated into osteoblast-like cells, but some infiltrating cells differentiated into bidirectional cells. When PPARγ was activated, differentiating GFP⁺Sca-1⁺/PDGFRα⁻ cells shifted from osteoblast-like to bidirectional cells or osteoclasts (<i>n</i> = 10 per group). *<i>P</i><0.005 compared to mice injected with BM-derived Sca-1⁺/PDGFRα⁻ cells. P, PPARγ agonist.</p

Vessel resident calcifying progenitor cells are mesenchymal but not hematopoietic.

<p>(A) FACS of arterial cells from GFP–BMT Apoe^−/− mice. GFP⁺ cells were negative for Lineage antibody cocktail targets. GFP⁺Lin⁻Sca-1⁺/PDGFRα⁺ or GFP⁺Lin⁻Sca-1⁺/PDGFRα⁻ cells expressed CD29 or CD106 (<i>n</i> = 10, performed in triplicate). (B) Schematic of osteoblast, adipocyte, and chondrocyte inductions from calcifying progenitor cells. Cells were stained with Alizarin Red S after 14 d of differentiation; bars: 1 mm. Oil Red O staining 28 d after differentiation; bars: 50 µm. Safranin O staining 28 d after differentiation; bars: 50 µm. (C) Adipocyte-related genes and chondrocyte-related genes were upregulated in Sca-1⁺ cells (Sca-1⁺/PDGFRα⁺, Sca-1⁺/PDGFRα⁻) under each differentiation condition.</p

Schematic figure of comparative mechanism of mDA neuron differentiation between mESC and P-iPSC.

<p>The SFRP1 level is the highest in undifferentiated cells. As differentiation begins, SFRP1 expression was decreased by specific methylation at 17^th, 18^th, 21^st, and 22^nd CpGs of its promoter. Epigenetically repressed SFRP1 gene fails to antagonize Wnt5a, which enables to augment Wnt5a signal transduction. Increased level of Wnt5a binds to the Frizzled (Fz) receptor and propagates dopamine neuron differentiating-signals by stimulating Lmx1a, b, Nurr1, Pitx3 and finally inducing tyrosine hydroxylase enzyme to specify neural precursor cells to mDA neurons.</p

In vitro characterization of pluripotency of mESCs and P-iPSCs and schematic overview of experimental design for mDA neuronal differentiation.

<p>(A) Confocal images of mESCs and P-iPSCs to show pluripotency markers, Oct-4 and SSEA1 within cell colonies. Nuclei were stained with Sytox blue. Oct-4, and SSEA1 were expressed in nucleus or membrane respectively. Scale bars  = 20 μm. (B) DA neuronal differentiation is composed of five stages. Each stage showed distinct morphological changes in stem cells. The aggregated form of EBs was attached on gelatin-coated dish for 7 days. During this step, cells were transformed into tightly-packed epithelial morphology in ITSFn media. Propagation of neuronal precursor cells begins when cultured in N3 media for 3 to 5 days. Lastly, terminal differentiation into mDA neuron begins after culturing them in N3 media for 8 to 10 days in the absence of bFGF and addition of ascorbic acid in N3 media for 8∼10 days.</p

The origin of calcifying progenitor cells.

<p>(A) BMT experimental outline. (B) After 5 d of cell infusion, we checked the presence of GFP+ cells in arteries and blood using FACS and immunofluorescent staining. A small fraction of donor GFP+ cells were detected in peripheral blood (1.5%) but were rarely detected in the artery (0.2%). Twelve weeks after transplantation GFP+ cells from donor BM were reconstituted with peripheral blood cells of C57 mice (up to 90%). At that time, 13% of arterial resident cells were GFP+. Thus, the majority of GFP+ cells gradually were incorporated into the artery within a considerable duration. Bars: 50 µm. (C) Aortas were harvested after 6 mo of BMT and were stained with antibodies targeting GFP, Sca-1, or PDGFRα. The three panels on the right depict high-magnification images of the white squares shown on the left. Blue, Sytox Blue nuclear staining; L, lumen; A, adventitia. White dashed lines describe the media. Bars: yellow = 100 µm; white = 20 µm. (D) Schematic of the GFP⁺ clonal expansion assay. (E) PI staining to identify fusion between GFP⁺ and non-GFP⁺ cells. (F) GFP⁺ single clone immunofluroscent staining. Bars: 200 µm. (G) Giemsa staining of single-cell colonies. (H) Osteocalcin/cathepsin K staining of osteoblast/osteoclast differentiation from GFP⁺ single colonies after 14 d.</p

Clonal expansion of calcifying progenitor cells.

<p>(A) Schematic of the clonal expansion assay. (B) Giemsa staining to detect a single-cell colony. Bars: 1 mm. (C) For statistical analysis, colony-forming cells were counted among 96 wells per group. Experiments were performed in triplicate. Colonies formed by Sca-1⁺ cells were much more compact and abundant than Sca-1⁻ cells. *<i>P</i><0.001 versus Sca-1⁻/PDGFRα⁺ cells. ‡<i>P</i><0.005 versus Sca-1⁺/PDGFRα⁺ cells. (D) Schematic depicting osteoblastic and osteoclastic differentiation of clonally expanded cells. (E) Alizarin Red S and osteocalcin staining to detect osteoblast differentiation from single-colony cells after 14 d of differentiation. Bars: black = 1 mm; white = 20 µm. (F) TRAP and cathepsin K staining to detect osteoclast differentiation from single-colony cells after 14 d of differentiation. Bars: black = 100 µm; white = 20 µm.</p

Osteoblastic and osteoclastic differentiation potentials of calcifying progenitor cells in the vasculature.

<p>(A) Sca-1 (red) and PDGFRα (green) immunostaining to detect vascular calcifying progenitor cells from WT C57 mouse aortas (<i>n</i> = 5). The three panels on the right depict high-magnification images of the white squares indicated on the left. The white dashed lines specify the culture media. L, lumen; A, adventitia; Blue, Sytox Blue nuclear staining. Bars: yellow = 100 µm; white = 20 µm. (B) Schematic of the experiments indicating the three different conditions of osteoblast induction. (C) Cells were cultured with 10% FBS; 10% FBS+10 ng/ml TNF-α; or 10% FBS+1.25 mM CaCl₂+2 mM β-glycerolphosphate. Compared with Sca-1⁻ cells, Sca-1⁺ cells cultured under osteoblastic conditions showed significantly higher numbers of ALP positive cells, greater ALP activity, and higher expression levels of osteoblast-related genes. *<i>P</i><0.01 versus Sca-1⁻/PDGFRα⁺ cells. Under moderate osteoblastic induction conditions (FBS only and FBS+CaCl₂), Sca-1⁺/PDGFRα⁻ cells expressed both RANK and RANKL. Under the most potent osteoblastic differentiation conditions (FBS+TNF-α), RANK expression was not induced. Experiments were performed in triplicate. (D) Schematic of the experiments of osteoclast induction. (E and F) Under osteoclastic differentiation conditions, Sca-1⁺/PDGFRα⁻ cells differentiated into TRAP-positive, multinucleated cells (>3 nuclei; <i>n</i> = 5 per group). *<i>P</i><0.001 versus Sca-1⁻/PDGFRα⁺ cells after 7 d of differentiation. Bars: 50 µm. (G) Osteoclast-related genes were upregulated in Sca-1⁺/PDGFRα⁻ cells. (H) Calcifying progenitor cells were cultured on calcium phosphate-coated discs. SEM imaging indicated that Sca-1⁺/PDGFRα⁻ cells generated a typical resorption area (calcium pore size) in contrast to Sca-1⁺/PDGFRα⁺ cells. Bars: 10 µm. (I) Under the same conditions in (H), Sca-1⁺/PDGFRα⁻ cells formed dual actin sealing zones as demonstrated by FITC-conjugated phalloidin staining. Bars: 50 µm.</p

The regulation of vascular calcification by calcifying progenitor cells.

<p>Illustration of calcifying/decalcifying progenitor cells and their proposed roles in noncalcified conditions (A) and atherosclerotic plaques (B). Sca-1⁺ progenitor cells do not express hematopoietic lineage markers but express MSC-like markers. Sca-1⁺/PDGFRα⁻ progenitor cells differentiate into osteoblasts/osteoclasts bidirectionally, whereas Sca-1⁺/PDGFRα⁺ progenitor cells are committed to osteoblastic fates. PPARγ activation can shift the direction of differentiation of Sca-1⁺/PDGFRα⁻ progenitor cells toward osteoclasts, but it cannot change the differentiation of Sca-1⁺/PDGFRα⁺ progenitors. (A) Under homeostatic noncalcified conditions, circulating BM-derived calcifying cells (Sca-1⁺ cells) infiltrated the adventitia through the vasa vasorum. (B) Under pathologic atherosclerotic calcification or therapeutic intravascular cell delivery, these cells might infiltrate into the intima directly from the blood.</p

Ex vivo BM-derived vascular calcifying progenitor cells modulated by PPARγ activation toward osteoblastic/osteoclastic differentiations.

<p>(A) Experimental outline (<i>n</i> = 10, performed in triplicate). (B and C) BM-derived GFP⁺ cells were treated with rosiglitazone under osteoblastic differentiation conditions (10% FBS+10 ng/ml TNF-α). ALP staining (B) indicated that PPARγ activation suppressed osteoblastic differentiation. TRAP staining (C) and RT–PCR revealed that PPARγ activation induced the osteoclastic differentiation of Sca-1⁺/PDGFRα⁻ cells. P, PPARγ agonist 1, 10, or 25 µM. Bars: 100 µm.</p