Data on the involvement of Meox1 in balloon-injury-induced neointima formation of rats

In the previous report, Meox1 was found to promote SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade (Wu et al., 2017) [1]. Here, we presented new original data on the involvement of Mesoderm/mesenchyme homeobox gene l (Meox1) in balloon-injury-induced neointima formation of rat. In rat carotid artery balloon injury model to induce vascular remodeling, Meox1 was induced in vascular smooth muscle cell (SMCs) of rat carotid arteries. Most proliferating cell nuclear antigen (PCNA)-positive cells also expressed Meox1. These data suggested that Meox1 may be involved in SMCs proliferation during injury-induced neointima formation. Furthermore, knocked down its expression in injured arteries by adenoviral delivery of Meox1 short hairpin RNA (shRNA) (shMeox1), neointima formation was significantly inhibited. Elastin staining also confirmed the reduction of neointima in Meox1 shRNA-transduced arteries. Moreover, knockdown of Meox1 decreased the collagen production/deposition that was significantly increased in neointima induced by balloon injury.


a b s t r a c t
In the previous report, Meox1 was found to promote SMCs phenotypic modulation and injury-induced vascular remodeling by regulating the FAK-ERK1/2-autophagy signaling cascade (Wu et al., 2017) [1]. Here, we presented new original data on the involvement of Mesoderm/mesenchyme homeobox gene l (Meox1) in balloon-injury-induced neointima formation of rat. In rat carotid artery balloon injury model to induce vascular remodeling, Meox1 was induced in vascular smooth muscle cell (SMCs) of rat carotid arteries. Most proliferating cell nuclear antigen (PCNA)-positive cells also expressed Meox1. These data suggested that Meox1 may be involved in SMCs proliferation during injury-induced neointima formation. Furthermore, knocked down its expression in injured Value of the data 1) The data revealed that Meox1 was induced in arterial SMCs during injury-induced vascular remodeling, and promoted injury-induced neointimal formation in rat carotid artery. 2) These data are the further evidence on the interplay between neointimal formation and Meox1 [1].
3) The data in this article provide the potential therapeutic effect of Ad-Sh-Meox1 on restenosis following percutaneous coronary intervention (PCI) through promoting re-endothelialization while inhibiting neointimal formation.

Data
To determine the role of Meox1 in SMCs phenotypic modulation in vivo, we used rat carotid artery balloon injury model to induce vascular remodeling. Injury induced progressive neointima formation in the arteries (Fig. 1A). Meox1 was initially induced in medial SMCs (1 and 3 days after the injury) and subsequently in neointimal SMCs (7-28 days after the injury) (Fig. 1A). To quantify Meox1 expression, we performed Western blot using proteins extracted from the sham-operated control or balloon-injured arteries. As shown in Fig. 1B-C, Meox1 was time-dependently induced along with a cell proliferating marker PCNA in carotid arteries following injury. To determine if Meox1 was expressed in SMCs and if Meox1-expressing cells contributed to the SMCs proliferation, we co-stained Meox1 with smooth muscle α-actin (α-SMA) and PCNA in arteries injured for 14 days, respectively. As shown in Fig. 1D, most Meox1-expressing cells were α-SMA-positive. Most PCNA-positive cells also expressed Meox1 (Fig. 1D).
To determine if Meox1 plays a role in the injury-induced neointimal formation, we knocked down its expression in injured arteries by adenoviral delivery of Meox1 shRNA. Knockdown of Meox1 ( Fig. 2A-B) significantly inhibited the neointima formation as well as the intima/media ratio by nearly 40% (Fig. 2C-D). Elastin staining also confirmed the reduction of neointima in Meox1 shRNA-transduced arteries. Moreover, knockdown of Meox1 decreased the collagen production/deposition that was significantly increased in neointima by balloon injury (Fig. 2E), which is likely due to the role of Meox1 in modulating SMCs phenotype in the neointima.

Rat carotid artery injury model
According to our previous protocals for model of balloon-injury carotid artery of rat [2], the mix gas including Isoflurane (1.5-2.5%) and oxygen was used for anesthetizing rats. After introducing a 2F Fogarty arterial embolectomy balloon catheter through the external carotid artery in left, pre-filled saline in the catheter were injected with 0.02 mL to inflate the balloon, and then rotatably withdrawn through the common carotid artery from proximal to the distal end of the heart. The above procedures were repeated for 3 times to achieve the perfect balloon-injury carotid artery with the traits of utter endothelial denudation. 100 µL of saline, Ad-Null or Ad-shMeox1 was injected into the ballooninjury carotid arteries through the specific syringe (WISP SYR, Hamilton), respectively. Subsequently, in order to transduce expression adenoviral vector of the interest genes into the cells of balloon-injury carotid artery, filling saline, Ad-Null or Ad-shMeox1 were resided in these arteries for incubation of 20 min, respectively.

Statistical analysis
All data were evaluated with a 2-tailed, unpaired Student t test or compared by 1-way ANOVA followed by the t-test and are expressed as mean 7SD. A value of P r0.05 was considered statistically significant.