Impaired angiogenesis during fracture healing in GPCR kinase 2 interacting protein-1 (GIT1) knock out mice

Abstract

G protein coupled receptor kinase 2 (GRK2) interacting protein-1 (GIT1), is a scaffold protein that plays an important role in angiogenesis and osteoclast activity. We have previously demonstrated that GIT1 knockout (GIT1 KO) mice have impaired angiogenesis and dysregulated osteoclast podosome formation leading to a reduction in the bone resorbing ability of these cells. Since both angiogenesis and osteoclast-mediated bone remodeling are involved in the fracture healing process, we hypothesized that GIT1 participates in the normal progression of repair following bone injury. In the present study, comparison of fracture healing in wild type (WT) and GIT1 KO mice revealed altered healing in mice with loss of GIT1 function. Alcian blue staining of fracture callus indicated a persistence of cartilagenous matrix in day 21 callus samples from GIT1 KO mice which was temporally correlated with increased type 2 collagen immunostaining. GIT1 KO mice also showed a decrease in chondrocyte proliferation and apoptosis at days 7 and 14, as determined by PCNA and TUNEL staining. Vascular microcomputed tomography analysis of callus samples at days 7, 14 and 21 revealed decreased blood vessel volume, number, and connection density in GIT1 KO mice compared to WT controls. Correlating with this, VEGF-A, phospho-VEGFR2 and PECAM1 (CD31) were decreased in GIT1 KO mice, indicating reduced angiogenesis with loss of GIT1. Finally, calluses from GIT1 KO mice displayed a reduced number of tartrate resistant acid phosphatase-positive osteoclasts at days 14 and 21. Collectively, these results indicate that GIT1 is an important signaling participant in fracture healing, with gene ablation leading to reduced callus vascularity and reduced osteoclast number in the healing callus.

Figure 1. GIT1 mRNA is expressed during fracture healing.

WT mice were administered femur fractures and fractured femora were harvested for isolation of mRNA from the callus at 7, 14, and 21 days post-injury. qPCR was performed to examine the profile of GIT1 expression. Bars represent mean GIT1 expression level relative to GAPDH +/− SEM (N = 4, *p<0.05).

Figure 2. Disjunction persists at 14 days post-fracture in GIT1 KO mice.

Femur fractures were induced in 10-week-old WT and GIT1 KO mice. Fractured femora were harvested for analysis at 7, 14, and 21 days post-injury. Radiographs obtained at the 14 day time point consistently revealed radiolucency in GIT1 KO calluses (B, red arrow) compared with calluses from WT mice (A, yellow arrow). This was supported by microCT analysis, which revealed lack of bridging mineral in GIT1 KOs (D, red arrows) compared to a connected shell of mineral in WT controls (C). Further quantification of callus geometry via microCT indicated that there were no differences in mineralized callus volume between WT and GIT1 KO mice (E). Bars represent mean callus volume (mm³) +/− SEM (N = 3, *p<0.05).

Figure 3. Cartilage persists and woven bone callus is delayed in GIT1 KO mice.

Histological analysis of fracture callus cartilage content was performed via Alcian Blue Hematoxylin/Orange G staining. Representative stains of calluses from WT and GIT1 KO mice at 1, 2 and 3 weeks post-fracture are displayed (A–F). Red arrows denote Alcian Blue-stained cartilagenous matrix and asterisks denote mineralized woven bone. Histomorphometry was performed on triplicate sections from multiple mice, with % Cartilage Area (G) and % Bone Area (H) quantified. Bars represent % Area (cartilage or bone) +/− SEM (*p<0.05, N = 3).

Figure 4. Type 2 collagen-containing matrix persists in GIT1 KO mice.

Tissue sections cut from WT and GIT1 KO mice were analyzed for COL2A1 content using an immunohistochemistry approach. Representative stains at 7, 14 and 21 days post-fracture are depicted, with asterisks denoting areas within the callus at 2 and 3 weeks post-fracture in GIT1 KO mice (D and F respectively) that have more robust/persistent staining.

Figure 5. Chondrocyte proliferation is reduced in GIT1 KO mice.

Representative PCNA staining is shown at 7 and 14 days post-fracture in WT mice (A and C) and at 7 (B) and 14 days (D) post-fracture in GIT1 KO mice (B and D respectively). Histomorphometry was performed on triplicate sections from multiple mice to quantify the number of PCNA-positive cells per unit callus area (E). The data is presented as mean of the number of PCNA positive cells/mm²+/− SEM (*p<0.05, N = 3).

Figure 6. Chondrocyte TUNEL staining is reduced in GIT1 KO mice.

Chondrocyte apoptosis was assessed in WT and GIT1 KO mice at 14 and 21 days post-fracture. Representative TUNEL immunofluorescence and DAPI staining at both time points is presented in WT mice (A/C and E/G respectively) and in GIT1 KO mice (B/D and F/H respectively). Quantitative histomorphometric analyses of the number of TUNEL-positive cells per unit area in triplicate sections from three WT and GIT1 KO mice at 14 and 21 days post-fracture are presented (I). Bars represent the percent of TUNEL positive cells/mm²+/− SEM (*p<0.05, N = 3).

Figure 7. Fracture callus vascularity is reduced in GIT1 KO mice.

To visualize and quantify callus vascularity, WT and GIT1 KO mice were perfused with lead chromate microfilm perfusion reagent. Harvested femora were decalcified and representative vascular microCT reconstructions from each experimental group at 7, 14 and 21 days post-fracture are presented. Reduced vascularity in GIT1 KO mice (A, C, E) compared to WT control mice (B, D, F) was evident at all time points. Quantification of callus vascular parameters, including Vessel Volume (G), Vessel Number (H), Vessel Spacing (I) and Connection Density (J) supported these findings, with GIT1 KO mice possessing reduced callus vessel volume, vessel number and connection density and increased space between vessels compared to callus from WT mice. Bars represent mean for each value +/− SEM (N = 3, *p<0.05).

Figure 8. PECAM1⁺ blood vessel number is reduced in GIT1 KO mice.

Representative PECAM1 immunofluorescence is presented at 7 and 14 days post-fracture in WT mice (A and C) and GIT1^−/− mice (B and D). Histomorphometry was performed to quantify the average number of positively-stained blood vessels present in each field of view on each section analyzed. Three sections (from 3 levels within each callus, 25–50 µm apart) were viewed using the 10× objective, with counts being collected from 3 fields of view in each section. All counts from each callus (9 fields total) were averaged. Vessel counting using this approach confirmed the immunofluorescence in panels A–D, with WT calluses possessing between 2 and 3-fold more PECAM1⁺ vessels than GIT1 KO calluses at both time points (E). Bars represent mean number of PECAM1⁺ vessels/field +/− SEM (*p<0.01, N = 3).

Figure 9. VEGF signaling is reduced in GIT1 KO mice.

Phospho-VEGF receptor immunostaining was performed on fracture calluses from WT and GIT1 KO mice at 2 and 3 weeks post-fracture. Panels A-D depict representative staining profiles, with Phospho-VEGF receptor-positive cells staining red as indicated by red arrows. Histomorphometry was performed to quantify the number of Phospho-VEGF receptor-positive cells per unit callus area (E). Data is presented as the mean number of positive cells per unit area (i.e. region of interest) +/− SEM (*p<0.01, N = 3). Additionally, immunohistochemistry was performed of assess VEGF levels in fracture calluses from WT (F) and GIT1 KO mice (G). Representative histological sections of calluses at 2 weeks post-fracture are presented, depicting reduced expression in KO mice. VEGF positive cells are stained reddish-brown as indicated by red arrows.

Figure 10. Osteoclast number is reduced in GIT1 KO mice.

The presence of osteoclasts in the fracture callus of WT and GIT1 KO mice was assessed at 7 (A and B respectively), 14 (C and D respectively) and 21 days (E and F respectively) post-fracture via TRAP staining. Histomorphometry to quantify percentage of osteoclast surface (G) and osteoclast number per unit bone surface (H) was also performed on triplicate sections from multiple mice, with bars representing the mean for each parameter +/− SEM (*p<0.05, N = 3).