A Comparative Study of ROCK Inhibitors Fasudil and Y27632 on Promoting Angiogenesis and Flap Survival in Mice

doi:10.12300/j.issn.1674-5817.2025.153

Abstract

Abstract:

Objective To compare the effects of the Rho-associated protein kinase (ROCK) inhibitors Fasudil and Y27632 on promoting angiogenesis in vivo and in vitro and on flap survival in mice. Methods Thirty-six SPF-grade adult male ICR mice were randomly divided into the Y27632 group, Fasudil group, and control group, with 12 mice in each group. From 3 d before surgery, the mice were intraperitoneally injected daily with Y27632 solution, Fasudil solution, and an equal volume of normal saline, respectively. Then, a peninsular flap model pedicled on the iliolumbar vessels on the back of the mice was established surgically. Laser speckle contrast imaging was used to evaluate flap blood perfusion immediately after surgery (day 0) and on day 5, and the pedicle artery diameter was measured and proximal and distal blood flow were calculated. On day 5 after surgery, the flap necrosis rate was calculated, and CD31-positive microvessel density in the distal part of the flap was detected by immunofluorescence. In addition, EdU cell proliferation, scratch, Transwell, and Matrigel tube formation assays were used to compare the effects of Fasudil and Y27632 on the proliferation, migration, invasion, and tube formation ability of human umbilical vein endothelial cells (HUVECs) in vitro, with DMSO treatment as the control. Results On day 5 after surgery, the flap necrosis rate in the Fasudil group was (2.0±1.5)%, which was significantly lower than that in the control group and the Y27632 group (P<0.001), and the necrosis rate in the Y27632 group was also significantly lower than that in the control group (P<0.001). The pedicle iliolumbar artery diameter in the Fasudil group on day 0 and day 5 after surgery was (143.8±4.2) μm and (177.6±7.6) μm, respectively, both significantly larger than those in the control group and the Y27632 group (P<0.001). On day 5 after surgery, the proximal and distal blood flow in the Fasudil group were (453.0±12.5) PU and (79.2±4.8) PU, respectively, both significantly higher than those in the control group(P<0.001) and the Y27632 group (P<0.05). The microvessel density in the distal surviving tissue of the flap was (71.6±3.5) vessels/region, significantly higher than that in the control group (P<0.001) and also significantly higher than that in the Y27632 group (P＜0.05). On day 0 after surgery, the pedicle iliolumbar artery diameter in the Y27632 group was smaller than that in the control group (P＜0.05), while on day 5 after surgery, there was no statistically significant difference between the two groups (P>0.05). The proximal and distal blood flow on day 5 after surgery were also significantly higher than that in the control group (P<0.001), and the microvessel density was also significantly higher than that in the control group (P<0.05), but the distal blood flow showed no significant difference compared with the control group (P>0.05). In vitro experiments showed that the proliferation rate, migration rate, and number of invasive cells of HUVECs in the Fasudil and Y27632 groups were significantly increased compared with the control group (P<0.001), but there was no statistically significant difference between the two drug groups (P>0.05), whereas the tube formation ability of the Fasudil group was significantly better than that of the Y27632 group (P<0.05). Conclusion Both Fasudil and Y27632 can effectively promote angiogenesis and improve flap survival rate, but the effect of Fasudil is more significant, which may be related to its sustained regulation of hemodynamics and vascular stability.

Key words: ROCK inhibitor, Blood perfusion, Angiogenesis, Flap survival, ICR mice

CLC Number:

R-332

ZHAN Shaoqin,FANG Fang,ZHUANG Yuehong,et al. A Comparative Study of ROCK Inhibitors Fasudil and Y27632 on Promoting Angiogenesis and Flap Survival in Mice[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 689-699. DOI: 10.12300/j.issn.1674-5817.2025.153.

Figures/Tables 6

Figure 1 Morphology of mouse skin flaps and skin flap necrosis rate on day 5 after surgery

Note： A-C， Flap morphology of the Y27632， Fasudil， and control groups on day 5 after surgery， respectively （mice were intraperitoneally injected with Y27632 solution， Fasudil solution， and an equal volume of normal saline for 3 days before surgery）. D， Statistical analysis showed that the flap necrosis rate in the Fasudil group was significantly lower than that in the Y27632 group and the control group， and the necrosis rate in the Y27632 group was also significantly lower than that in the control group. n=6 mice per group，^*** P<0.001.

Figure 2 Changes in the pedicle iliolumbar artery diameter and blood perfusion of mouse flaps

Note： A， Laser speckle contrast imaging showing flap blood perfusion immediately after flap harvesting （recorded as day 0）and on day 5 in each group （mice were intraperitoneally injected with Y27632 solution， Fasudil solution and an equal volume of normal saline， respectively， for 3 consecutive days before surgery）. B， Quantitative analysis of the pedicle iliolumbar artery diameter， proximal flap perfusion （PU， perfusion unit）， and distal flap perfusion at day 0 after surgery. C， Quantitative analysis of the corresponding indicators at day 5 after surgery. PU， perfusion units. n = 6 mice per group. ^ns P > 0.05，^** P < 0.01， ^*** P < 0.001.

Figure 3 CD31 immunofluorescence staining of skin flap tissues in mice on day 5 after surgery

Note： A–C， CD31 immunofluorescence staining of microvessels in the distal surviving tissue of the flaps in the Y27632， Fasudil， and control groups， respectively （mice were intraperitoneally injected with Y27632 solution， Fasudil solution， and an equal volume of normal saline for 3 days before surgery）. Scale bar = 250 μm. D， Quantitative analysis of the mean microvessel number per field in the distal surviving tissue of the flap in each group. n=6 in each group， ^* P<0.05， ^*** P<0.001.

Figure 4 Comparison of proliferation of human umbilical vein endothelial cells in vitro in each group

Note： A， Representative fluorescence images of the EdU proliferation assay （scale bar=250 μm）； B， Quantitative analysis of the proportion of EdU-positive cells among the three groups （^ns P>0.05， ^*** P<0.001）. Cells in the Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO for 6 h， respectively， and labeled with 10 μmol/L EdU reagent， with 3 wells per group. EdU， 5-ethynyl-2'-deoxyuridine；DAPI， 4'，6-diamidino-2-phenylindole.

Figure 5 Comparison of the migration ability of human umbilical vein endothelial cells in vitro in each group

Note： A-B， Horizontal and vertical migration abilities of HUVECs measured by the plate scratch assay and Transwell assay respectively （the scale bar is 100 μm）； C-D， Statistics of cell migration rate （i.e.， the proportion of scratch healing area） and the number of cells penetrating the polycarbonate membrane respectively （^ns P>0.05， ^*** P<0.001）. Cells in the Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO， respectively， with 3 wells per group.

Figure 6 Comparison of the tube formation ability of human umbilical vein endothelial cells in vitro in each group

Note： A， Matrigel tube formation assay was used to detect the in vitro tube-forming ability of HUVECs in each group （scale bar = 100 μm）； B-E， Quantitative analysis of the tube formation assay showing the total length of master segments， the number of master segments， the number of master junctions where two or more tubular segments intersect in the tubular structure， and the number of master meshes. The Y27632， Fasudil， and control groups were treated with Y27632 solution （10 μmol/L）， Fasudil solution （10 μmol/L）， and an equal volume of DMSO， respectively， with 3 wells per group，^*** P<0.001.

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