Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: PDGFRβ+ cells are perivascular cells (PDGFRβ+ PVC) but are distinct from mature pericytes in tumours. (a) Blood vessels in pancreatic tumours were visualized with FITC-labelled tomato lectin (Lycopersicon esculentum) that was injected intravenously into 13-week-old Rip1Tag2 mice prior to killing them. Tumour sections were then stained with a red-labelled anti-PDGFRβ antibody. PDGFRβ+ cells are in close adjunction to blood vessels (white arrowhead) and can bridge between blood vessels (red arrowhead). (b–d) Immunohistochemical analysis of PDGFRβ+ cells24 and mature pericytes (red). Tumour sections were co-stained with anti-PDGFRβ24 and anti-desmin (b), anti-αSMA (c) or anti-NG2 (d) to reveal colocalization. Predominantly, PDGFRβ+ cells were distinct from mature pericytes (white arrowheads), but expression overlapped in a few areas (yellow arrowheads)20. (e) Quantification of NG2+/PDGFRβ+ cell populations in Rip1Tag2 pancreatic tumours. Tumours were dispersed into single cells, incubated with antibodies for PDGFRβ24 and NG2 (red) and then sorted by FAC. Three cell populations were revealed: 46% expressed PDGFRβ but not NG2, 26% were immunoreactive for both PDGFRβ and NG2, while 28% only expressed NG2. αSMA+ and desmin+ pericytes could not be isolated by FACS due to the nature of the commercially available antibodies. Scale bars, 8.7 mm.

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: Injection, Staining, Immunohistochemical staining, Expressing, Incubation, Isolation

Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: PDGFRβ+ cells differentiate into mature pericytes in vitro. (a–f) PDGFRβ+ cells were isolated from tumours with a green fluorescent PDGFRβ antibody that was coated to magnetic beads and then cultured in vitro. Cells were immunolabelled with antibodies for the mature pericyte markers NG2 (a, b), αSMA (c, d) or desmin (e, f) immediately after the cells had settled onto the dish (day 0; a, c, e) or after 7 days in culture (day 7; b, d, f). (g) PDGFRβ+ cells that were positive for either NG2, αSMA or desmin, were counted at day 0 and day 7 to reveal the induction of mature pericytes. Whereas NG2+ and αSMA+ cells increased about fourfold after 7-day culture when compared with freshly isolated cells, desmin+ cells did not expand in number during the cell culture. Addition of the cell-cycle blocker mitomycin-C (5–10 μg ml−1; 7dM) during culture did not change the ratio of pericyte differentiation. (h) Quantitative Taqman RT–PCR analysis for RGS-5, a marker of developing and angiogenic pericytes, was performed on total RNA isolated from tumour-derived PDGFRβ+ (freshly isolated and cultured for 7 days) and NG2+ cells in Rip1Tag2 tumours. Expression levels were normalized to those of L19. RGS-5 levels were very high in isolated PDGFRβ+ cells (day 0) but markedly reduced in differentiated PDGFRβ+ cells in culture (day 7), and in mature tumour-derived NG2+ pericytes. Scale bars, 9 mm.

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: In Vitro, Isolation, Magnetic Beads, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Marker, Derivative Assay, Expressing

Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: PDGFRβ+ cells and endothelial cells in co-culture form pericyte-covered vascular tubes. (A) Time-lapse imagings of co-cultured endothelial cells24 and PDGFRβ+ cells (red) in 3D Matrigel at different time points. Human microvascular endothelial cells (HDMECs) were labelled with a green fluorescent vital dye and PDGFRβ+ cells, isolated from pancreatic tumours, with a red fluorescent vital dye and co-cultured in a 3D Matrigel. Vessel assembly was observed with a confocal microscope over a period of 18 h using time-lapse imaging. HDMECs were also cultured with the pancreatic islet tumour cells βTC3 over 18 h but these cells remained randomly distributed in the Matrigel (A, e). (B) Endothelial cell and PDGFRβ+ cell co-cultures form complex vascular tubes that are covered with mature periyctes leading to the induction of all three pericyte markers: NG2, desmin and αSMA. Endothelial cells24 and PDGFRβ+ cells (unlabelled) were co-cultured in Matrigel and the induction of NG2 (a–d), αSMA (e–h) and desmin (i–l) was visualized with red-labelled antibodies for the pericyte markers after 3 h (a, e, i) and 7 days in co-culture in the presence (b, f, j) or absence of TGFβ activity (c, g, k). Mature pericytes (red) elongate and wrap around endothelial tubes24 as indicated by white arrowheads. Endothelial tubes form a vessel lumen (yellow arrowheads). The increase in mature pericytes at 1-, 3- and 7-day cultures was quantified by comparing the ratio of pericytes at 1, 3 and 7 days to the numbers of cells at 3 h of incubation (d, h, l). In the presence of neutralizing TGFβ antibodies, αSMA+ cells, but not NG2+ or desmin+ cells, were reduced by 40% in a 7-day culture (*P = 0.0066). Scale bars, 2.3 mm (A), 5 mm (B, a, e, i), 14.9 mm (B, b, f, j), 9.9 mm (B, c, g, k).

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: Co-Culture Assay, Cell Culture, Isolation, Microscopy, Imaging, Activity Assay, Incubation

Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: Tumour-associated PDGFRβ+ PPCs originate from bone-marrow-derived haematopoietic Sca1+ cells. (a) Quantification of Sca1+/PDGFRβ+ cell populations in Rip1Tag2 pancreatic tumours. Tumours were dispersed into single cells, incubated with antibodies for PDGFRβ and Sca1 (red) and then sorted by FACS. Three populations were revealed: 67% expressed PDGFRβ but not Sca1, 19% were immunoreactive for both Sca1 and NG2, while 17% expressed only Sca1. (b, c) Sca1+ cells (red) in normal exocrine pancreas (b) and in pancreatic islet tumours (c). The vasculature is visualized in green with FITC–tomato lectin. Higher magnifications in the boxes visualize Sca1+ cells within blood vessels in normal pancreas (b; red arrowhead), whereas numerous Sca1+ cells were observed in close association with tumour vessels in the perivascular space (c; red arrowhead). (d) Tumour sections of irradiated Rip1Tag2 mice that were reconstituted with bone marrow cells from syngeneic actin–GFP mice. Bone-marrow-derived cells are visualized in green, the vasculature was labelled with rhodamine–lectin and changed to false colour blue, and NG2+ cells were detected with an antibody for NG2 in red. Yellow arrowhead points to a GFP–NG2 double-positive cell. FACS analysis of GFP–bone-marrow-reconstituted tumours reveals GFP cells that are positive for PDGFRβ and/or NG2. (f) Quantitative analysis of GFP cells expressing PDGFRβ and/or NG2. (g–i) A subset of GFP cells in tumours (g) coexpress Sca1 (h) and NG2 (i). (j–o) Tumour- (j) and bone marrow (k–o)-isolated Sca1+ cells were co-cultured with HDMEC endothelial cells in a 3D Matrigel (j–m) or as monolayers on plastic dishes (n, o). The induction of NG2 (j, k, m), αSMA (l, o) and desmin (n) was visualized with red-labelled antibodies for the pericyte markers after 5 days in co-culture. Either isolated Sca1+ cells were co-cultured with pre-labelled HDMECs (green vital dye) (j–l), or Sca1+ cells were pre-labelled in green and cultured with unlabelled HDMEC (m–o). The latter allowed the identification of double-positive cells by their combined green and red colour when Sca1+ cells expressed a mature pericyte marker (m–o; yellow arrowheads). Sca1+ cells induced expression of NG2 (j, k, m), αSMA (l, n) and, to a lesser extent, desmin (o) after 5 days of culture. Scale bars, 7 mm (g–i).

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: Derivative Assay, Incubation, Irradiation, Expressing, Isolation, Cell Culture, Co-Culture Assay, Marker

Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: Inhibition of PDGFRβ signalling depletes pericytes and increases endothelial cell apoptosis. (A) Anti-PDGFRβ treatment in immunocompromised Rip1Tag2-Rag1ko/ko mice depletes tumour pericytes. Ten-week-old Rip1Tag2-Rag1ko/ko mice (n = 8) were subjected to rat anti-mouse PDGFRβ or saline (control) for 3 weeks. Mice were injected intravenously with FITC-labelled tomato lectin prior to sacrifice to visualize the vasculature in green. Subsequently, tumour sections of control and treated mice were stained with a red-labelled antibody for PDGFRβ (a, b), desmin (d, e), αSMA (g, h) or NG2 (j, k). In contrast to control tumours, very few pericytes were observed in treated tumours (red arrowheads). Pericyte-depleted blood vessels were enlarged and hyperdilated with vessels in control tumours (white arrowheads). Quantitative evaluation of the number of PDGFRβ+ (c), desmin+ (f), αSMA+ (i) and NG2+ (l) cells was revealed on control and treated tumour sections by red antibody staining. The total area of red staining within the tumour boundaries within each image (7–13 images per set) was quantified using Improvision’s Volocity 2.6.1. Statistical analysis was performed with an unpaired t-test comparing the pericyte coverage of control-treated to anti-PDGFRβ tumours. P values were considered statistically strongly significant (P < 0.01).

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: Inhibition, Injection, Staining

Journal:

Article Title: PDGFR? + perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival

doi: 10.1038/ncb1288

Figure Lengend Snippet: PDGFRβ+ cells support vascular tube stability and survival. (a, b) Human microvascular endothelial cells (HDMECs) were labelled with a green fluorescent vital dye and cultured in a 3D Matrigel in the absence (a) or presence (b) of tumour-isolated PDGFRβ+ cells (pre-labelled in red). Vessel assembly occurred quickly in both situations, but bare endothelial tubes started to deteriorate after 2 days in culture resulting in endothelial cell clumps after 7 days (a). In contrast, PDGFRβ+ cell-covered tubes (white arrowheads) remained intact even after 7 days (b). (c) Quantitative RT–PCR analysis of VEGF from total RNA of tumour-isolated PDGFRβ+ cells, NG2+ cells and from total Rip1Tag2 tumours (RT2). VEGF transcription levels were normalized to levels of L19. VEGF transcription levels are highly enriched in PDGFRβ+ cells and NG2+ pericytes compared with VEGF levels in Rip1Tag2 tumours. Scale bars, 12.6 mm.

Article Snippet: Pericytes were identified using the following antibodies: mouse anti-human desmin (1:100; DAKO Corp., Carpinteria, CA), mouse anti-human smooth muscle actin (1:500; DAKO Corp.), rabbit anti-mouse NG2 (1:500; Chemicon, Temecula, CA), mouse anti-human CD140b/PDGFRβ (1:200, BD Biosciences Pharmingen, San Diego, CA), rat anti-mouse PDGFRβ (1:100, eBiosciences, San Diego, CA), rabbit anti-mouse PDGFRβ 40 and R-Phycoerytherin (RPE)-conjugated rat anti-mouse Sca1 (1:200; BD Biosciences Pharmingen).

Techniques: Cell Culture, Isolation, Quantitative RT-PCR

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Preclinical evaluation of 68 Ga-radiolabeled trimeric affibody for PDGFRβ-targeting PET imaging of hepatocellular carcinoma

doi: 10.1007/s00259-023-06260-x

Figure Lengend Snippet: a The transcriptomic profile of typically upregulated angiogenesis-associated genes in tumor tissues derived from HCC patients. b Protein expression profiles of angiogenesis-associated genes that were correlated with the severity of HCC. c H&E and PDGFRβ staining of liver tissues derived from HCC patients or residual normal liver tissues of the donor liver for transplantation. PDGFRβ-positive cells (red) were visualized by antibodies against human PDGFRβ. The nuclei of the cells were stained by DAPI (blue). d Western blot of PDGFRβ in normal or HCC tissues ( n = 3)

Article Snippet: To detect the colocalization of PDGFRβ and Z TRI in tumor vessels, HCC tissues were sectioned as 6-μm slices under frozen conditions, followed by incubation with goat anti-human PDGFRβ (AF385; R&D, MN), rabbit anti-rat PDGFRβ (380,772; ZenBio, Chengdu, China), or rat anti-human CD31 (303,102, BioLegend, CA; Clone:WM59) at 37 °C for 1.5 h. Subsequently, the tissues were washed with PBS and incubated with corresponding secondary antibodies (BioLegend, CA) for an additional 0.5 h prior to observation under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Germany).

Techniques: Derivative Assay, Expressing, Staining, Transplantation Assay, Western Blot

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Preclinical evaluation of 68 Ga-radiolabeled trimeric affibody for PDGFRβ-targeting PET imaging of hepatocellular carcinoma

doi: 10.1007/s00259-023-06260-x

Figure Lengend Snippet: Z TRI shows high affinity for PDGFRβ. a Affinity of Z MON and Z TR affibody to PDGFRβ analyzed by biolayer interferometry at different concentrations. b Binding of Z TRI to PDGFRβ. + pericyte was evaluated by flow cytometry. c Co-localization of Z TRI (green) with PDGFRβ (red) in normal and HCC clinical specimens. The nuclei of cells were visualized by DAPI staining (blue)

Article Snippet: To detect the colocalization of PDGFRβ and Z TRI in tumor vessels, HCC tissues were sectioned as 6-μm slices under frozen conditions, followed by incubation with goat anti-human PDGFRβ (AF385; R&D, MN), rabbit anti-rat PDGFRβ (380,772; ZenBio, Chengdu, China), or rat anti-human CD31 (303,102, BioLegend, CA; Clone:WM59) at 37 °C for 1.5 h. Subsequently, the tissues were washed with PBS and incubated with corresponding secondary antibodies (BioLegend, CA) for an additional 0.5 h prior to observation under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Germany).

Techniques: Binding Assay, Flow Cytometry, Staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Preclinical evaluation of 68 Ga-radiolabeled trimeric affibody for PDGFRβ-targeting PET imaging of hepatocellular carcinoma

doi: 10.1007/s00259-023-06260-x

Figure Lengend Snippet: a Schematic illustration for the preparation of [ 68 Ga]Ga-DOTA-Z TRI . Z TRI affibody was conjugated with DOTA-NHS ester followed by radiolabeling with 68 Ga. b Stability of [ 68 Ga]Ga-DOTA-Z TRI in PBS and FBS for 6 h in vitro. c Cellular binding assay of [ 68 Ga]Ga-DOTA-Z TRI to PDGFRβ + pericytes. d Autoradiography of [ 68 Ga]Ga-DOTA-Z TRI and PDGFRβ immunofluorescent staining in human liver tissues with or without HCC

Article Snippet: To detect the colocalization of PDGFRβ and Z TRI in tumor vessels, HCC tissues were sectioned as 6-μm slices under frozen conditions, followed by incubation with goat anti-human PDGFRβ (AF385; R&D, MN), rabbit anti-rat PDGFRβ (380,772; ZenBio, Chengdu, China), or rat anti-human CD31 (303,102, BioLegend, CA; Clone:WM59) at 37 °C for 1.5 h. Subsequently, the tissues were washed with PBS and incubated with corresponding secondary antibodies (BioLegend, CA) for an additional 0.5 h prior to observation under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Germany).

Techniques: Radioactivity, In Vitro, Cell Binding Assay, Autoradiography, Staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Preclinical evaluation of 68 Ga-radiolabeled trimeric affibody for PDGFRβ-targeting PET imaging of hepatocellular carcinoma

doi: 10.1007/s00259-023-06260-x

Figure Lengend Snippet: a Biodistribution of [ 68 Ga]Ga-DOTA-Z TRI in BALB/c mice. Mice were intravenously injected with 740 kBq of [ 68 Ga]Ga-DOTA-Z TRI followed by collection the majority organs at 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h post-injection ( N = 5). Red arrow points out the necrotic lesion and the yellow arrow points out the nodule lesion. b PET/CT imaging of [ 68 Ga]Ga-DOTA-Z TRI in DEN-induced primary HCC rats and healthy ones. Rats were intravenously injected with 5.55 MBq of [ 68 Ga]Ga-DOTA-Z TRI and scanned at 1 h post-injection. Tumor lesion was pointed out by red arrows. The corresponding liver tissues were collected and [ 68 Ga]Ga-DOTA-Z TRI uptake was investigated by autoradiography after scanning. H&E staining was used to identify the structure of tumor tissues, while AFP immunohistochemical staining and PDGFRβ immunofluorescent staining were performed as index of HCC

Article Snippet: To detect the colocalization of PDGFRβ and Z TRI in tumor vessels, HCC tissues were sectioned as 6-μm slices under frozen conditions, followed by incubation with goat anti-human PDGFRβ (AF385; R&D, MN), rabbit anti-rat PDGFRβ (380,772; ZenBio, Chengdu, China), or rat anti-human CD31 (303,102, BioLegend, CA; Clone:WM59) at 37 °C for 1.5 h. Subsequently, the tissues were washed with PBS and incubated with corresponding secondary antibodies (BioLegend, CA) for an additional 0.5 h prior to observation under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Germany).

Techniques: Injection, Positron Emission Tomography-Computed Tomography, Imaging, Autoradiography, Staining, Immunohistochemical staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: Preclinical evaluation of 68 Ga-radiolabeled trimeric affibody for PDGFRβ-targeting PET imaging of hepatocellular carcinoma

doi: 10.1007/s00259-023-06260-x

Figure Lengend Snippet: PET/CT imaging of [ 68 Ga]Ga-DOTA-Z TRI in rhesus monkeys. a Representative PET imaging of rhesus monkey with primary HCC. Monkeys were intravenously injected with a dose of 3.7 MBq/kg of [ 68 Ga]Ga-DOTA-Z TRI , [ 68 Ga]Ga-DOTA-Z MON , or [ 18 F]-FDG, respectively. Tumor lesion is indicated by red arrows. b Paired comparison of HCC monkey and healthy monkey. Uptake of [ 68 Ga]Ga-DOTA-Z TRI in liver and tumor were evaluated using fused PET/CT imaging and autoradiography. H&E staining and AFP immunohistochemical staining were performed to identify whether the tissue is cancerous. Co-localization of Z TRI (green) with PDGFRβ (red) in collected liver tissues was verified by immunofluorescent staining. The nuclei of cells were visualized by DAPI staining (blue)

Article Snippet: To detect the colocalization of PDGFRβ and Z TRI in tumor vessels, HCC tissues were sectioned as 6-μm slices under frozen conditions, followed by incubation with goat anti-human PDGFRβ (AF385; R&D, MN), rabbit anti-rat PDGFRβ (380,772; ZenBio, Chengdu, China), or rat anti-human CD31 (303,102, BioLegend, CA; Clone:WM59) at 37 °C for 1.5 h. Subsequently, the tissues were washed with PBS and incubated with corresponding secondary antibodies (BioLegend, CA) for an additional 0.5 h prior to observation under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Germany).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Injection, Comparison, Autoradiography, Staining, Immunohistochemical staining

Journal: Brain Pathology

Article Title: Denser brain capillary network with preserved pericytes in Alzheimer's disease

doi: 10.1111/bpa.12897

Figure Lengend Snippet: Pericyte density changes in AD . A. Representative confocal image showing PDGFRβ‐immunoreactive pericytes and the laminin‐immunoreactive vascular basement membrane in the frontal cortex of an AD case. The arrowhead demonstrates a PDGFRβ‐immunoreactive pericyte body contained within the laminin‐immunoreactive basement membrane. Those were counted with the stereological fractionator method (see Supporting Figure for details). B. The density of pericytes (number of pericytes in a given tissue volume) is increased in AD cases (one‐way ANOVA; * P = 0.00058). C and D. The linear density of pericytes (pericyte number in a given capillary segment; C; one‐way ANOVA; P = 0.46) as well as the vascular coverage ( D ; Mann–Whitney U test; P = 0.78) did not change in AD cases compared to controls. Scale bar: 50 µm. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: For the stereological measurement of pericyte linear density and proliferation, the following primary antibodies were employed: laminin (mouse anti‐human, γ1, clone 2E8, Millipore, 1:800); PDGFRβ (rabbit anti‐human, Y92, GeneTex, 1:100); Ki67 (Abcam, 1:1000).

Techniques: MANN-WHITNEY

Journal: International Journal of Molecular Medicine

Article Title: Total flavonoids of Rhizoma Drynariae enhances CD31 hi Emcn hi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF-BB/VEGF/RUNX2/OSX signaling axis

doi: 10.3892/ijmm.2022.5167

Figure Lengend Snippet: TFRD promotes CD31 hi Emcn hi vessel formation in angiogenic-osteogenic coupling during distraction osteogenesis via the PDGF-BB/PDGFR-β pathway. (A) Representative immunofluorescence images and (B) quantification of RUNX2, OSX, CD31 and PDGF-BB in the distracted tibias after distraction for 4 weeks. (C) Representative western blotting images and (D) semi-quantitative analyses of PDGF-BB, VEGF, RUNX2, OSX as well as the phosphorylation of AKT and ERK1/2 in the distracted tibias at 4 weeks post-distraction. (E) Quantification of mRNA expression levels of PDGF-BB, VEGF, RUNX2 and OSX. Data represent the mean ± SD. n=3 rats in each group from three independent experiments. *P<0.05 vs. control group; # P<0.05 vs. PDGF-BB-Ab group. TFRD, total flavonoids of Rhizoma drynariae ; PDGF, platelet-derived growth factor; Emcn, endomucin; PDGFR-β, platelet-derived growth factor receptor-β; RUNX2, runt-related transcription factor 2; OSX, Osterix; p-, phosphorylated.

Article Snippet: Subsequently, the membranes were blocked with 5% (w/v) non-fat dry milk in TBS with 0.05% Tween-20 (Beyotime Institute of Biotechnology) at room temperature for 45 min, followed by incubation with primary anti-PDGF-BB (1:500; cat. no. ab21234; Abcam), anti-VEGF (1:500; cat. no. sc-7269; Santa Cruz Biotechnology, Inc.), anti-RUNX2 (1:1,000; cat. no. ab23981; Abcam), anti-OSX (1:1,000; cat. no. ab22552; Abcam), anti-phosphorylated (p)-AKT (1:1,000; cat. no. ab81283; Abcam), p-PDGFR-β (1:2,000; cat. no. AF3132; Affinity Biosciences), PDGFR-β (1:2,000; cat. no. AF6133; Affinity Biosciences), AKT (1:1,000; cat. no. 4691; Cell Signaling Technology, Inc.), ERK (1:2,000; cat. no. AF0155; Affinity Biosciences) and anti-p-ERK1/2 (1:1,000; cat. no. ab214362; Abcam) antibodies at 4°C overnight.

Techniques: Immunofluorescence, Western Blot, Expressing, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Total flavonoids of Rhizoma Drynariae enhances CD31 hi Emcn hi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF-BB/VEGF/RUNX2/OSX signaling axis

doi: 10.3892/ijmm.2022.5167

Figure Lengend Snippet: Schematic diagram of the underlying mechanism of TFRD-regulated CD31 hi Emcn hi vessel formation and subsequent bone regeneration during DO. TFRD, total flavonoids of Rhizoma drynariae ; PDGF, platelet-derived growth factor; RUNX2, runt-related transcription factor 2; OSX, Osterix; Emcn, endomucin; PDGFR-β, platelet-derived growth factor receptor-β; DO, distraction osteogenesis.

Article Snippet: Subsequently, the membranes were blocked with 5% (w/v) non-fat dry milk in TBS with 0.05% Tween-20 (Beyotime Institute of Biotechnology) at room temperature for 45 min, followed by incubation with primary anti-PDGF-BB (1:500; cat. no. ab21234; Abcam), anti-VEGF (1:500; cat. no. sc-7269; Santa Cruz Biotechnology, Inc.), anti-RUNX2 (1:1,000; cat. no. ab23981; Abcam), anti-OSX (1:1,000; cat. no. ab22552; Abcam), anti-phosphorylated (p)-AKT (1:1,000; cat. no. ab81283; Abcam), p-PDGFR-β (1:2,000; cat. no. AF3132; Affinity Biosciences), PDGFR-β (1:2,000; cat. no. AF6133; Affinity Biosciences), AKT (1:1,000; cat. no. 4691; Cell Signaling Technology, Inc.), ERK (1:2,000; cat. no. AF0155; Affinity Biosciences) and anti-p-ERK1/2 (1:1,000; cat. no. ab214362; Abcam) antibodies at 4°C overnight.

Techniques: Derivative Assay