Sonic Hedgehog Improved Endothelial Cell Dysfunction and Attenuated The Development of Atherosclerosis In Mice

Sonic hedgehog (Shh), an evolutionally-highly conserved morphological factor whose maturation, transportation and function were closely related to cholesterol. Shh played an important role in maintaining adult coronary vasculature homeostasis. It not only induced angiogenesis to improve myocardial infarction but also inhibited ox-LDL induced endothelial apoptosis. However, the role of Shh in endothelial cell injury have not been fully elucidated. Here, we shown Shh induced nitric oxide (NO) release and endothelial nitric oxide synthase (eNOS) synthesis, which improved endothelial cell dysfunction and inhibited atherosclerotic plaque. In vivo, Shh reduced the plaque lesion in high fat diet (HFD) induced ApoE -/- mice. In endothelial cell, Shh improved NO and eNOS mRNA expression and inhibited intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) mRNA expression. In contrast, knockdown of Shh inhibited eNOS and NO level and induced ICAM-1 and VCAM-1. In conclusion, we found that Shh has anti-inammation and improved endothelial cell injury consequently attenuated the development of atherosclerosis.


Introduction
Endothelial cell dysfunction was regarded as the crucial and early change in the life history of atherosclerotic lesion and was a key factor to the underlying pathobiology of atherosclerotic cardiovascular disease (ASCVD) [1]. Changes in vascular endothelium, broadly speaking, included a range of modulations in functional phenotypes, vascular tension, redox balance and in ammation, which provided a vital link to atherosclerotic lesion initiation and progression [2]. Multiple factors such as hyperlipidemia, hypertension, hyperglycemia and smoking derived circulating low density lipoprotein (LDL) accumulation in the arterial lesion-prone zone and LDL oxidation modi cation into oxidized low density lipoprotein (ox-LDL), which resulted in augment of reactive oxygen species (ROS), inhibition of the endothelial nitric oxide synthase (eNOS) synthesis and nitric oxide (NO) release, subsequently, elevated expression of cell surface adhesion factors including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), recruiting of circulating monocytes to the intima, induced a complex process of endothelial cell dysfunction and launched pathogenic occlusion [3]. Focusing on the causes and courses of endothelial cell dysfunction and inhibiting them have been an important therapeutic target for ASCVD.
Sonic hedgehog (Shh) was a highly evolutionarily conserved morphogen which could shape into concentration gradients to provide cells location information and determine cell fates [4]. Shh morphogen served as an important role in the development and homeostasis maintenance of blood vessels in embryo and postnatal and abnormal expression of Shh was associated with various birth defects and tumors. As previously studies, Shh established their morphogen gradients nothing but covalently modi ed by cholesterol and palmitoyl groups [5]. Shh lacking cholesterol resulted in the change of subcellular distribution and seriously disrupted the organization structure [6]. Functional Shh contained cholesterol and palmitoyl hydrophobic moieties which was manifestly incompatible with long-distance functions. Drosophila hedgehog could be carried by the insect lipophorin which strongly resemble mammalian LDL, and lipophorin-bound hedgehog was capacity to transport to target cells [7]. A set of studies demonstrated that ox-LDL was strongly associated with progression of atherosclerosis, thusly, further investigating the relationship between ox-LDL and Shh is with large unmet medical needs.
In this study, we addressed the potential role of Shh in ox-LDL induced endothelial cell dysfunction and atherosclerosis. In vivo, Shh attenuated the action of high fat diet (HFD) on the atherosclerosis plaque area in ApoE −/− mice. And also, Shh improved NO and eNOS expression. In vitro, upon ox-LDL treatment,

Animal Model
Male ApoE −/− mice aged 8 weeks old on a C57BL/6 background were obtained from Model Animal Research Center of Nanjing University, China. All mice were kept on a standard lighting (12 h light/12 h dark cycle), constant temperature (22-24℃), and humidity (50-60%). Experiment mice were given a High Fat Diet (HFD). We randomly divided the mice into three groups for intravenous injection treatment (n=20 in each group): Lv-Shh group (lentivirus at a dose of 2*10 7 TU/mouse), Sh-Shh group (lentivirus at a dose of 2*10 7 TU/mouse), and negative group. At the end of 12 weeks, all the mice were euthanized for subsequent study. The animal care and procedures were complied with the guidelines of animal welfare.

Tissue preparation
The whole aortas and hearts were rapidly removed and washed in PBS. In en face oil red O staining, the adventitia of aortas was identi ed by oil red O. In en face oil red O staining, the adventitia of aortas was stripped at rst, then the aortic arches were dissected.

Cell culture
HUVECs were obtained from human umbilical cord veins treated with a 0.25% trypsin solution following previously published methods. Cells were cultured in Endothelial Cell Medium (ScienCell Research Laboratories, Inc.) consisting of basal medium, 5% FBS, 1% EC growth supplement and 1% penicillin/streptomycin solution in a humidi ed incubator at 37℃ in a 5% CO 2 atmosphere. Cells were used between passages 3 to 5.

RNA isolation and real-time PCR assay
Total RNA was extracted using TRlzol regent (Invitrogen, Thermo Fisher Scienti c, Inc.). The total RNA (1000 ng) was subjected to a reverse transcription reaction with the PrimeScriot TM room temperature (RT) Reagent Kit (Takara, Japan). Real-PCR was carried out using Sybr Premix Ex Taq TM II (Takara, Japan). Table 1 The primer sequences for the qRT-PCR

Species
Gene

Statistical analysis
All statistical analysis was performed using GraphPad Prism 8.0. The data were presented as the mean ± standard deviation (SD), and one-way ANOVA was used to perform statistical comparisons followed by Tukey's post hoc test. P < 0.05 was considered to indicate a statistically signi cant difference.

Effect of Shh in ApoE −/− mice in the atherosclerosis
To test the effect of Shh in HFD induced ApoE −/− mice, we operated by intravenous injection lentivirus including Lentivirus Shh and Shh shRNA by tail. Firstly, we veri ed that Shh gene transfer was successfully in aortic tissue ( Figure 1A). Furthermore, we compared the mice artery plaque area after 12 weeks. As shown in Figure 1B, HFD induced the formation of atherosclerotic plaque especially in aorta, carotid artery and abdominal aorta, and compared with the negative control, lentivirus Shh could reduce the area of plaque, in contrast, Shh shRNA increased the plaque. It was interesting to note there was no signi cant difference among the negative control group, lentivirus Shh group and Shh shRNA group in weight and cholesterol including T-CHO, TG, LDL-C and HDL-C ( Figure 1C). Furthermore, to assess the Shh effect on endothelium function, we measured the expression of NO in serum and eNOS mRNA and protein in aortic tissue. Lentivirus Shh induced the expression of NO in serum and eNOS mRNA and protein in aortic tissue, but Shh shRNA was inversive ( Figure 1D). These results suggested that Shh inhibited endothelial cell dysfunction and attenuated the development of atherosclerosis in ApoE −/− mice.

Effect of Shh in endothelial cell dysfunction in ox-LDL-Induced HUVECs
It was reported that ox-LDL was an inducing factor in endothelial dysfunction [8]. In this study, we used HUVECs transfected with plasmid encoding Shh or pretreated with recombinant Shh protein followed by a 24 h co-incubation with ox-LDL (50 µg/mL) to observe the effect of Shh on endothelial dysfunction in vitro. Ox-LDL induced reactive oxygen in HUVECs, resulting in an increase in ROS level which is consistent with the previous results [9]. Furthermore, transfected with plasmid encoding Shh effectively suppressed the production of ROS (Figure 2A). Consistent with the results of plasmid encoding Shh gene, ROS level decreased in a dose-dependent manner after recombinant Shh protein administration ( Figure 2B). In other hand, ox-LDL treatment induced signi cant downregulation of NO level, which was known as an effective endogenous vasodilator [10]. As shown in Figure 2C, plasmid encoding Shh gene could induced NO expression. Alike, compared with ox-LDL group, recombinant Shh protein motivated the releasing of serum NO level ( Figure 2D). In keeping with NO expression, plasmid and recombinant protein of Shh induced eNOS mRNA expression ( Figure 2E and F). These results shown that Shh improved endothelial cell dysfunction by downregulation of ROS and promoting the NO and eNOS expression.

Effect of Shh on in ammation in ox-LDL-Induced HUVECs
According to the protein-protein interaction in STRING network, we found that eNOS had relationship with ICAM-1 and VCAM-1 which members of cell adhesion molecules ( Figure 3A). As former studies have proved cell adhesion molecules as in ammation factor accelerated endothelial cell dysfunction. We veri ed the effect of Shh on ICAM-1 and VCAM-1 in response to endothelial cell dysfunction. As shown in Figure 3B and C, ox-LDL treatment had a notable enhancement of ICAM-1 and VCAM-1 expression. Pretreatment with plasmid encoding Shh gene or recombinant Shh protin effectively suppressed the production of ICAM-1 and VCAM-1 mRNA level.

Silencing Shh induced endothelial cell dysfunction and in ammation
To further clarify Shh improved endothelial cell dysfunction and inhibited in ammatory response, we used siRNA to knockdown Shh. As shown in Figure 4A, siRNA 1, 2, 3 could interfere Shh mRNA expression and siRNA 2 was most remarkable, so we chosen siRNA2 for forward study. Transfected with Shh siRNA alone could induced the expression of ROS and ox-LDL in development the ROS level and could be weaken by recombinant Shh protein ( Figure 4B). For another, ox-LDL treatment induced signi cant downregulation of NO level which was suppressed by recombinant Shh protein, and to the contrary, accelerated by knockdown of Shh ( Figure 4C). Consistent with NO expression, siRNA inhibited eNOS mRNA expression ( Figure 4D). Further, Shh siRNA could induced the expression of ICAM-1 and VCAM-1 and recombinant Shh protein could inhibit siRNA induced ICAM-1 and VCAM-1 expression to some extent ( Figure 4E). Therefore, we demonstrated that silencing Shh could induce endothelial cell dysfunction and may rescue by recombinant Shh protein.

Discussion
Improving of endothelial cell dysfunction was a vital step in suppression the progression of atherosclerosis and preventing acute cardiovascular events, which bring out massive medical burden globally [11]. Therefore, it was of great signi cance to actively explore the molecular mechanism of atherosclerosis in the prevention and treatment of cardiovascular diseases and related complications. In preview study, we had identi ed that Shh expression was suppressed in ox-LDL-induced HUVECs and in addition, Shh could improve endothelial apoptosis. In this study, we found that Shh could promote NO and eNOS expression, leading to a signi cant suppression of atherosclerotic plaques in ApoE −/− mice.
Consistent with these data, Shh could improve endothelial cell dysfunction by promoting expression of NO and eNOS and inhibiting expression of ROS and in ammation in ox-LDL-induced HUVECs. These results suggest that Shh was a promising therapeutic target for atherosclerosis.
Endothelial cell dysfunction is a major pathogenetic contributor of early-stage atherosclerotic lesions which is characterized by endothelial cells maladapted including reducing of eNOS and NO expression and inducing ROS expression, resulting in promoting endothelial cells activated and in ammation whereas vasoconstriction [12]. Under uncontrolled uptake of ox-LDL, impaired release of cholesterol, and excessive cholesterol esteri cation could result in accumulation of cholesterol esters stored as cytoplasmic lipid droplets and subsequently trigger endothelial dysfunction. Control of the homeostasis of endothelium has a critical importance in the pathogenesis of atherosclerosis, we observed that Shh exerted a protective effect by down-regulation ROS and promoted eNOS and NO releasing. Furthermore, we undertook an in vivo demonstrated that Shh could reduce the plaque lesion in HFD-diet ApoE −/− mice. These results provide strong evidence to support the notion that Shh contributed to a protective effect through increased eNOS and NO expression, as well as inhibited tra cking of cell surface molecules, thereby exerted anti-arteriosclerosis. Moreover, previous study has found that Shh could reduce the endothelial cell apoptosis associated with the NF-κB signaling. This remind us that this pathway may also be involved in the regulation of the endothelial cell dysfunction. Further work is needed before a de nitive conclusion on this matter can be draw.
In recent years, scholars have found that Shh has neuroprotective effects on anti-oxidative stress and anti-apoptosis [13]. In the cortical neuron damage induced by H 2 O 2 , Shh could promote the expression of anti-apoptotic gene Bcl-2 and inhibit the expression of pro-apoptotic gene Bax, and simultaneously upregulate neurotrophic factor, vascular endothelial growth factor and brain-derived neurotrophic factor to protect cortical neurons from oxidative stress [14]. In addition, Shh increased the activities of superoxide dismutase and glutathione peroxidase, and up-regulates the expression of Bcl-2 protein, which improved the ability of autistic patients to defend against oxidative stress [15]. In previous study, in ox-LDL-induced HUVECs apoptosis model, we also found that Shh, as a protective protein, can inhibit the mitochondriamediated apoptosis. And further, in this study we found that Shh have anti-in ammation and endothelial relaxing.
In short, these data suggest that Shh may attenuate ox-LDL induced endothelial cell dysfunction through