Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

Abstract

Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki-67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

Figure 1

hVW-MSC aspect and HGF/c-MET expression. (a) hVW-MSCs were enzymatically isolated from human arteries and, at passage 3, they exhibited adherence to the plastic substrate and a typical spindle-shaped morphology. Scale bars = 100 μm. (b) Flow cytometry analysis of mesenchymal, stemness, pericyte, and hematopoietic and vascular markers expressed in our cell model of hVW-MSCs. Values are reported in percentage of positivity. (c) Basal expression of HGF (252 bp) and c-MET (440 bp) mRNA in hVW-MSCs, detected by RT-PCR. (d) c-Met and HGF protein detection on cell lysates of hVW-MSCs exposed to HGF/SF (0, 2.5, 5, and 10 ng/mL) for 24 hrs.

Figure 2

Proliferative effect of HGF/SF on hVW-MSCs. (a) In vitro proliferation of hVW-MSCs exposed to HGF/SF assessed by Alamar Blue fluorescence assay up to 7 days. From day 1 to day 3, the fluorescence intensity was increased in HGF/SF-treated hVW-MSCs including untreated cells (red) while no difference was seen between hVW-MSCs stimulated by HGF/SF and untreated cells at 3 and 7 days. (b) Representative images of cycling cells expressing Ki-67 protein after 6 and 24 hrs of incubation with HGF/SF, compared to controls (hVW-MSCs). Ki-67 positive cells (green) and DAPI (nuclei in blue). After 6 hrs, the HGF/SF stimulation induced a slight increase of Ki-67 positive hVW-MSCs percentage on total DAPI cells; after 24 hrs of HGF/SF exposure, the percentage of Ki-67 positive cells is approximately reduced of the 15% in all the experimental conditions. Scale bars = 50 μm. (c) Images and quantitative analysis of hVW-MSCs cultured with PHA-665752 and then exposed to HGF/SF. c-Met inhibitor before HGF/SF incubation revealed almost similar Ki-67 percentage reduced in comparison to control cells. Scale bars = 50 μm. (b)-(c) ^∗, ^∗∗, ^∗∗∗ p value < 0.05, and one-way ANOVA test followed by Bonferroni posttest.

Figure 3

Migratory effect of HGF/SF on hVW-MSC. (a) Crystal Violet staining of hVW-MSCs migrated into the scratch area after 24 hrs of exposure to HGF/SF in comparison to the untreated hVW-MSCs; the cell migration was slackened by PHA-665752 before treatment. Wounded area is delimited by black lines. Scale bars = 50 μm. (b) Quantitative analysis of cell migration area in hVW-MSCs treated with HGF/SF, in presence and absence of PHA-665752. (c) Immunofluorescence staining for Vimentin revealed that the intermediate filaments Vimentin (positive (green)) was markedly stained in migrated cells (arrows) then unmigrated cells. Scale bars = 100 μm. The reported images are representative of three independent experiments.

Figure 4

Motility effect of HGF/SF on hVW-MSCs. Representative Crystal Violet and SEM images of (a) untreated (hVW-MSCs) and (b) HGF/SF- (10 ng/mL) treated hVW-MSCs migrated through an 8 μm porous membrane adhering to the migration surface. The cell-seeding surface analysis showed that the chemotactic HGF/SF facilitated the migration of many hVW-MSCs. Histological and SEM images: scale bars = 50 μm.

Figure 5

Angiogenic effect of HGF/SF on hVW-MSCs. (a) Representative in vitro tube-like formation images of HGF/SF (10 ng/mL for 7 days) and VEGF (50 ng/mL for 7 days) preconditioned hWV-MSCs as well as uninduced hWV-MSCs and HUVEC in Matrigel assay for 2, 6, and 24 hrs from seeding. Scale bars = 100 μm. (b) The quantitative analysis of the capillary-like structures revealed a higher number of hVW-MSCs exposed to HGF/SF than VEGF-treated cells. ^∗ p value < 0.05; Student's t-test. (c) Flow cytometry analysis of von Willebrand factor (vWF), KDR, and CD31 expression on untreated hVW-MSCs, HUVEC, and HGF/SF- and VEGF-treated cells. (d) The angiogenic lineage commitment was evaluated for each treatment attributing a score from 0 to 4 according to flow cytometry values.