Thrombospondin-4 (TSP4) gene-modified bone marrow stromal cells (BMSCs) promote the effect of therapeutic angiogenesis in critical limb ischemia (CLI) of diabetic rats

Background: Critical limb ischemia (CLI) is the leading cause of lower limb amputation. Traditional treatments for CLI have limitations. Studies have shown that thrombospondin-4 (TSP4) can promote the growth of neovascularization. Results: In this study, we observed the angiogenesis efficiency of TSP4-overexpressing BMSC transplantation in CLI treatment. The recombinant FT106-tsp4-gfp lentiviral vector plasmid was constructed and transfected into 293FT cells. Primary BMSCs were successfully infected with the tsp4 virus, and TSP4 overexpression was confirmed before TSP4-BMSCs infusion. In vitro, TSP4-BMSCs were co-cultured with human umbilical vein endothelial cells (HUVECs). Vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β) secretion were measured in the co-culture supernatants by ELISA. The effect of TSP4-BMSCs on endothelial cell proliferation and migration was detected. Meanwhile, the effects of TSP4-BMSC on the angiogenesis of endothelial cells were tested by tube formation experiment and arterial ring test. In vivo, a rat CLI model was established, and 60 CLI rats were randomly divided into the CLI, BMSC + CLI and TSP4-BMSC + CLI groups. The effect of TSP4-BMSC on angiogenesis was detected by the motor function, immunohistochemistry and immunofluorescence staining assays. Neovascular density was detected by digital substraction angiography (DSA). Our results demonstrated that TSP4-BMSCs obviously increased TSP4, VEGF, Ang-1, MMP9, MMP2 and p-Cdc42/Rac1 expression in endothelial cells. TSP4-BMSCs treatment notably upregulated the TGF-β/smad2/3 signal pathway in HUVECs. In vivo, TSP4-BMSCs improved the motor function score of the CLI rats and increased MMP2, MMP9, Ang-1,

Critical limb ischemia (CLI) is the most advanced stage of arteriosclerosis obliterans quantity and low efficiency of angiogenesis, which limit its clinical efficacy in the treatment of CLI.

3.
Thrombospondin-4 (TSP4) is a member of the thrombospondin family that includes 4 other proteins (TSP1, TSP2, TSP3, and TSP5) [9], which are new proangiogenic extracellular matrix (ECM) proteins. TSP4 influences multiple extracellular responses in vitro that translate into enhanced blood vessel formation in vivo [10]. Therefore, in this study, we evaluated whether TSP4 overexpression in BMSCs could promote angiogenesis and further improve the efficacy of BMSC transplantation for CLI treatment.

Arterial ring experiment
The thoracic cavity of adult rats was exposed using surgical tools. The thoracic aorta was isolated, cut into sections of approximately 1 mm and embedded in 48well plates coated with Matrigel. The three groups of cell culture supernatants were added to each well, and duplicate wells were set for each group. The cells were cultured in a cell culture incubator, and images were taken using an inverted phase contrast microscope (Axio Observer 3, Carl Zeiss AG) at 72 h. The density of new blood vessels was measured using image analysis software (Image-Pro Plus Version 6.0, USA)

Wound Healing and Tube Formation Assays
Cultured HUVECs were used to evaluate cell migration with the wound healing assay. During the HUVEC logarithmic growth period, 6-well plates containing confluent cells were scratched in a straight line with a pipette tip to simulate a wound; then, the HUVECs were rinsed 3 times with PBS. Subsequently, the cells were incubated with the three groups of media continuously for 24 h, and wound closure was observed. Photographs were captured at 0 and 24 h with a phasecontrast microscope (Olympus, Japan). Simultaneously, HUVECs were grown in 96well plates coated with Matrigel, followed by incubation with conditioned media from the three groups for 24 h. The cells were photographed using a phase-contrast microscope, and the number of branch points was counted to verify the capacity for angiogenesis in each group. The Image-Pro Plus software was applied to quantitatively analyse the density of new blood vessels. were expressed as the mean ± standard deviation (SD). Differences were considered to be statistically significant at p < 0.05.

Characterization of cultured BMSCs
As illustrated in Fig. 1a, from passage 2, the cells became uniform and grew in whirlpool, radial, or parallel patterns. As shown in supplemental The percentage of gfp-positive cells was 65.49%±0.0145, which showed that the infection was successful (Fig. 1e). The Western blotting results showed that TSP4 expression was obviously higher in the TSP4-BMSCs than in the normal BMSCs (**p < 0.01) ( Fig. 1f-g). Fluorescent staining indicated that the TSP4 protein was expressed in the TSP4-BMSCs and was mainly present in the cytoplasm (Fig. 1h).
The ELISA results showed that significantly more TSP4 and VEGF proteins were secreted into the cell supernatant compared to that of normal BMSCs (**p < 0.01) ( Fig. 1i-j). The above results indicated that tsp4 gene fragments were inserted into the BMSCs and that the TSP4 protein was expressed both intra-and extracellularly.

TSP4-BMSCs promoted HUVEC migration and angiogenesis
The ELISA data showed that more TSP4 was secreted by HUVECs in the TSP4-BMSC group than in the BMSC group (*p < 0.05). TSP4 and TGF-β secretion by the HUVECs was also increased by BMSC treatment compared to that of the control group (*p < 0.05). Furthermore, the VEGF, TGF-β and TSP4 levels were increased following incubation of the TSP4-BMSCs with HUVECs compared with that of the control group at 48 h (**p < 0.01, *p < 0.05) (Fig. 2a-c). To elucidate the role of TSP4-BMSCs in the induction of angiogenesis, we performed the arterial ring and tube formation assays in cultured endothelial cells. The quantified results for tube length and tubular branch points showed that angiogenesis was significantly improved by TSP4-BMSC treatment compared with those of the control group at 48 h (**p < 0.01). The circumference of the arterial ring in the TSP4-BMSC group was also significantly longer than that of the BMSC group (*p < 0.01). (Fig. 5d-g).
Because the proliferation and migration of HUVECs are the key processes involved in angiogenesis, we also assessed whether the TSP4-BMSCs mediated proliferation and migration using the wound healing test. The results showed that the TSP4-BMSCs significantly increased HUVEC proliferation and migration in the control group at 24 h (**p < 0.01) (Fig. 2 h-i).

TSP4-BMSCs enhanced the expression of angiogenic factors in HUVECs
Western blotting analysis was used to quantify the protein expression levels of angiogenic factors derived from HUVECs incubated with conditioned medium at 48 h. Fig. 3a-b showed that TSP4 expression was significantly higher in the TSP4-BMSC group than in the control and BMSC groups (**p < 0.01). Treatment with the TSP4-BMSCs significantly increased VEGF, Ang-1, MMP9 and MMP2 expression compared with treatment with BMSCs alone and the control (**p < 0.01). Treatment with TSP4-BMSCs also markedly enhanced phosphorylated Cdc42/Rac1 expression compared with that of the other two groups after 48 h of incubation (**p < 0.01) ( Fig. 3a-b). The above results illustrated that TSP4-BMSCs might have an additive effect on improvement of angiogenic factor expression in HUVECs.

5.
TSP4-BMSCs activated the TGF-β/smad2/3 signalling pathway in HUVECs tendency continued to the 28th day. In addition, the motor function scores in the TSP4-BMSC + CLI group were notably higher than those in the CLI group at 7, 14 and 28 days (**p < 0.01,* p < 0.05). These data indicated that TSP4-BMSC treatment promoted motor function recovery.

7.
TSP4-BMSCs increased angiogenesis in the ischaemic areas of the rats Our immunohistochemical staining shown in Fig. 5a-f indicated that MMP2, MMP9 and Ang-1 expression was more distinct in the TSP4-BMSC + CLI group than in the other groups (**p < 0.01) and that expression in the BMSC + CLI group was significantly higher than that in the CLI group (**p < 0.01). As an important angiogenic factor, VEGF plays a primary role in facilitating angiogenesis. The quantitative analysis of the fluorescent immunostaining images suggested that VEGF expression was more significant in the TSP4-BMSC + CLI group than in the BMSC + CLI and CLI groups (**p < 0.01) and that BMSC treatment obviously increased VEGF expression compared with that of the CLI group (**p < 0.01) ( Fig.   5g-h). As a marker of endothelial cells, vWF expression was more distinguishable in the TSP4-BMSC + CLI group than in the other groups (**p < 0.01), and expression in the BMSC + CLI group was notably higher than that in the CLI group in our immunofluorescence assay (**p < 0.01) ( Fig. 5i-j). These results illustrated that TSP4-BMSC treatment dramatically increased VEGF and vWF expression in the ischaemic area. The data of DSA shown that vascular density and blood supply area near the ischemic area in the TSP4-BMSC + CLI group was significantly higher than that in the CLI group (**p < 0.01,* p < 0.05) (Fig. 6).

1.
Critical limb ischemia (CLI) is the leading cause of lower limb amputation. Most CLI patients suffer from diabetes or chronic renal failure or both [12]. Increased plasminogen activator inhibitor and von Willebrand factor in the blood circulation of diabetic patients leads to the increase of fibrinase activity and the decrease of prostaglandin I2 (PGI2), which leads to the increase of vasoconstriction/diastolic dysfunction and platelet adhesion and aggregation, thus triggering the occurrence of thrombosis in diabetic patients [13]. In addition, hyperglycemia in the blood circulation leads to an increase in terminal glycosylation products, which easily binds to specific receptors on vascular endothelium, leading to endothelial dysfunction [14]. Vascular endothelial cells can synthesize and secrete vasoactive substances, such as ET-1 and NO, to regulate the contractile/diastolic function of vascular smooth muscle. The terminal glycosylation product bind to vascular endothelial cells, resulting in dysfunction of endothelial cell secretion, imbalance of NO and ET-1 secretion, inhibition of active factors in vascular wall and circulating blood, and make the hypercoagulability of circulatory system [14]. Specifically, diabetic CLI patients should undergo revascularization immediately because the 5-year survival rate in these patients is reported to be as low as 25%, and diabetes is associated with increased risk of amputation and repeat revascularization procedures [12].
Conventional endovascular treatment suggests balloon angioplasty or bare metal stenting as a rescue strategy in the case of residual stenosis or flow-limiting dissection. However, diabetes mellitus and chronic renal failure contribute to the formation of aggressive, hard atherosclerotic plaques with marked calcifications that are resistant to balloon dilation, reducing the possibility of adequate acute lumen dilation with conventional balloon angioplasty [12].

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Isner et al. [15] reported the efficacy of gene therapy in using vascular endothelial growth factor plasmids to promote angiogenesis, and thus therapeutic angiogenesis has potential prospects for CLI patients. Potential cell therapies are based on the stimulation of angiogenesis with extracellular or cellular components, including endothelial progenitor cells and stem cells [16][17][18]. Most of these therapies use bone  The results showed that TSP4-BMSC treatment promoted the recovery of motor function in the rats. Immunohistochemistry and immunofluorescence staining were used to assess the expression of cytokines in ischaemic muscle tissue from the rats.
The results showed that TSP4-BMSC treatment significantly promoted MMP2, MMP9, Ang-1, vWF and VEGF expression in the rat ischaemic muscle tissue. Meanwhile, new blood vessels can be observed around the ischemic area after TSP4-BMSCs treatment. The above results indicated that TSP4-BMSCs promoted angiogenesis of ischaemic muscle tissue in rats, which was related to vascular growth factor secretion.

Supplementary Files
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