Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: A. Illustration of OPN isoform primary domain structure. Each block corresponds to an exon (numbered). OPNa is full length (top), OPNb lacks exon 5 (middle), and OPNc lacks exon 4 (bottom). Expanded amino acid sequences of exons 4 and 5, absent in OPNc and OPNb, respectively, are included. B. OPN primers were used to measure OPNa (277bp), OPNb (235bp), and OPNc (196bp) mRNA levels in tissue samples from non-ischemic tissues and tissues from PAD patients with critical limb ischemia by RT-PCR (n=3–5). Isoform plasmid DNA controls and beta actin for loading are both shown. C. To investigate if OPN isoforms differentially affect collateral vessel formation in vivo , the hindlimb ischemia was performed on the following groups: WT, OPN −/− , or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. Perfusion was measured by Laser Doppler perfusion imaging (LDPI). Representative LDPI images 14 days post-HLI are shown. D. Ischemic limb (IL) perfusion was quantified and normalized to the contralateral non-ischemic limb (NIL) and compared across groups. * p<0.05, † p<0.001 vs. GFP; d14, n=6. E. To determine if the OPN isoform effects on perfusion translate to increased functional limb use, animals were given free access to a running wheel (d7 post-HLI) and allowed to run for 7 days. Total distance run (meters) by was plotted for all groups as a measure of limb function. *p<0.05, † p<0.001 vs. GFP; n=9–10.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, In Vivo, Imaging, Functional Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: To determine if OPN isoforms differentially affect arteriogenesis, tissue sections from animals 14 days post-HLI treated (trx) with lentivirus (LV) to overexpress OPN isoform a, b, or c were stained with α smooth muscle actin (α-SMA). α-SMA positive vessel numbers and sizes were quantified as a readout for arteriogenesis. A. The number of α-SMA positive vessels was counted across treatment groups and plotted (p = ns). B. α-SMA positive vessel sizes were measured in WT or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. The number of vessels measured within the arteriole (10 – 200 μm 2 ), small artery (200 – 700 μm 2 ) and large artery (1000 – 2500 μm 2 ) size ranges were compared across all animal groups. Data are expressed as % change compared to +LV-GFP (control). *p<0.05 vs. OPNa, † p<0.001 vs. OPNb; n=8–10. C. Representative histology images from 14 days post-HLI stained with α-SMA are shown. α-SMA stain is red and counterstain is violet. Scale bars = 500 μm.

Article Snippet: For macrophage polarization studies, 3 hours after plating cells were stimulated with 10% FBS-RPMI with 100 ng/mL purified recombinant human OPNa, OPNb, or OPNc (cat# TP304803, TP305118, and TP31059; Origene, Rockville, MD), or were stimulated with 20 ng/mL of either interferon gamma (INFγ) or interleukin (IL)-4 for 48 hours (cat# 554587 and 550067; BD Biosciences) as positive controls for macrophage polarization.

Techniques: Staining

Journal: Bone Research

Article Title: Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

doi: 10.1038/s41413-021-00150-4

Figure Lengend Snippet: SPP1 and CXCL12 restored angiogenesis under inflammation in vitro. a Real-time qPCR analyses were performed to determine the relative expression of Spp1 and Cxcl12 in 10 dpf fracture calluses from control and RA mice ( n = 4). The mRNA levels were normalized to that of Actb and then normalized to the control group. * P < 0.05 compared with control by Student’s t test. b Immunohistochemical staining of 10 dpf fracture calluses from control and RA mice for SPP1 and CXCL12. c Representative images from HUVEC migration and tube formation assays using culture medium from vehicle- and IL-1β-treated chondrocytes supplemented with SPP1 and CXCL12, respectively. Quantification of ( d ) HUVEC migration as well as ( e ) tube number and tube length ( n = 3). All results were normalized to the controls. * P < 0.05 compared with control by two-way ANOVA. Scale bar, 200 μm

Article Snippet: The growth factors 100 μg·mL −1 SPP1 (Peprotec, #120-35) and 20 μg·mL −1 CXCL12 (Peprotec, #300-28B) were mixed with 0.5% gelatin (Millipore Sigma, #G6650) in syringe pump C. PCL was injected at a rate of 5 mL·h −1 onto the collecting mandrel 20 cm away.

Techniques: In Vitro, Expressing, Control, Immunohistochemical staining, Staining, Migration

Journal: Bone Research

Article Title: Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

doi: 10.1038/s41413-021-00150-4

Figure Lengend Snippet: PCL scaffolds gradually released SPP1 and CXCL12 in vitro. a Schematic illustration of the simultaneous electrospinning and electrospraying system for PCL scaffold fabrication. b Representative confocal images of the PLGA microspheres encapsulated with growth factors. PLGA: red; growth factor: green. c Representative SEM images of the PCL scaffold. d Profiles of SPP1 and CXCL12 release from PCL scaffolds loaded with SPP1 and CXCL12 ( n = 5). Scale bar, 200 μm

Article Snippet: The growth factors 100 μg·mL −1 SPP1 (Peprotec, #120-35) and 20 μg·mL −1 CXCL12 (Peprotec, #300-28B) were mixed with 0.5% gelatin (Millipore Sigma, #G6650) in syringe pump C. PCL was injected at a rate of 5 mL·h −1 onto the collecting mandrel 20 cm away.

Techniques: In Vitro

Journal: Bone Research

Article Title: Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

doi: 10.1038/s41413-021-00150-4

Figure Lengend Snippet: The release of SPP1 and CXCL12 promoted angiogenesis under inflammatory conditions. a Schematic illustration of the collagen construct that was used to create the 3D cell culture environment to examine the impact of released growth factors on HUVEC migration and tube formation. b Quantification of HUVEC numbers at different depths in collagen gel ( n = 5). All results were normalized to the controls. * P < 0.05 compared with control by two-way ANOVA. c Representative images of HUVEC lumen formation in collagen gels. d Quantification of lumen density at different depths in the collagen gel ( n = 5). * P < 0.05 compared with control by two-way ANOVA. Scale bar, 200 μm

Article Snippet: The growth factors 100 μg·mL −1 SPP1 (Peprotec, #120-35) and 20 μg·mL −1 CXCL12 (Peprotec, #300-28B) were mixed with 0.5% gelatin (Millipore Sigma, #G6650) in syringe pump C. PCL was injected at a rate of 5 mL·h −1 onto the collecting mandrel 20 cm away.

Techniques: Construct, Cell Culture, Migration, Control

Journal: Bone Research

Article Title: Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

doi: 10.1038/s41413-021-00150-4

Figure Lengend Snippet: The controlled release of SPP1 and CXCL12 restored angiogenesis and fracture nonunion in RA mice. a PCL scaffold with or without SPP1 and CXCL12 was applied to the fractured bone in RA mice. b MicroCT assessment of newly formed vessels within fracture calluses from RA mice treated with scaffolds with or without SPP1 and CXCL12 ( n = 5) at 10 dpf. c Quantification of the vessels in RA fracture calluses ( n = 5) at 10 dpf based on microCT assessment. The results were normalized to the scaffold only group. * P < 0.05 compared with control by Student’s t- test. d Immunohistochemical staining for endomucin in fracture calluses from RA mice at 10 dpf. e Quantification of the vessels in RA fracture calluses at 10 dpf ( n = 5) based on the immunohistochemical assessment. The results were normalized to the scaffold only group. * P < 0.05 compared with control by Student’s t test. f Representa t ive ABH/OG staining of fracture callus sections from RA mice treated with scaffold with or without SPP1 and CXCL12 at 10 dpf ( n = 5). g Histomorphometric quantification of bone area was performed on 10 dpf fracture callus sections from RA mice treated with scaffolds with or without SPP1 and CXCL12 ( n = 5). The results were normalized to the scaffold group. h Biomechanical torsion testing of RA fractures treated with scaffold with or without SPP1 and CXCL12 at 28 dpf ( n = 8). Max torque and displacement at max were quantified. All results were normalized to the controls. * P < 0.05 compared with control by Student’s t test. Scale bar, 200 μm

Article Snippet: The growth factors 100 μg·mL −1 SPP1 (Peprotec, #120-35) and 20 μg·mL −1 CXCL12 (Peprotec, #300-28B) were mixed with 0.5% gelatin (Millipore Sigma, #G6650) in syringe pump C. PCL was injected at a rate of 5 mL·h −1 onto the collecting mandrel 20 cm away.

Techniques: Control, Immunohistochemical staining, Staining

Journal: Bone Research

Article Title: Targeting angiogenesis for fracture nonunion treatment in inflammatory disease

doi: 10.1038/s41413-021-00150-4

Figure Lengend Snippet: Primer sequences for qPCR

Article Snippet: The growth factors 100 μg·mL −1 SPP1 (Peprotec, #120-35) and 20 μg·mL −1 CXCL12 (Peprotec, #300-28B) were mixed with 0.5% gelatin (Millipore Sigma, #G6650) in syringe pump C. PCL was injected at a rate of 5 mL·h −1 onto the collecting mandrel 20 cm away.

Techniques: