Coronary heart disease (CHD) is the most common type of cardiovascular diseases caused by atherosclerosis. Therefore it is of great importance to determine the risk factors and underlying mechanisms associated with CHD. Diabetes is considered to be one of the major risk factors for CHD, even after adjusting for the effects of hypertension, age, and smoking [1]. Moreover, diabetes mellitus is currently exhibiting an epidemic trend worldwide [2]. A retrospective cohort study by Booth et al. [3] showed that patients with type 2 diabetes mellitus (T2DM) had a 2- to 4-fold higher risk of developing CHD compared to the general population, and that the mortality rate of CHD was also increased [4]. Following percutaneous coronary intervention (PCI), the risk of stent restenosis in CHD patients with T2DM was 2.5-fold higher than those without T2DM [5].

Retinol-binding protein 4 (RBP4) is a novel adipokine secreted by adipocytes and the liver, and is significantly elevated in patients with T2DM [6]. Previous studies have confirmed that RBP4 can be involved in the development of T2DM by inducing insulin resistance and impairing islet $\beta$-cell function [7, 8]. A prospective cohort study by Liu et al. [9] found a close association between RBP4 and CHD. The study showed the expression of RBP4 increased in aortic atherosclerosis in both humans and mice and RBP4 tended to localize in regions rich in macrophage foam cells. This study also showed that baseline RBP4 levels remained an independent predictor for adverse cardiovascular events, even after adjustment for traditional risk factors [9].

These findings on the correlation between RBP4, T2DM and CHD suggest that elevated RBP4 levels act as an important risk factor for the progression of coronary lesions in patients with CHD combined with T2DM [10]. As an integral part of the cardiovascular system, changes in arterial elasticity often occur in the early stages of the disease and could reflect dysfunction of the entire cardiovascular system. Arterial elasticity has been shown to be an important predictor for the development of cardiovascular disease [11, 12]. There are currently several tools available for the measurement of coronary artery elasticity function. Intravascular ultrasound (IVUS), an invasive examination, is able to obtain images of lumen changes inside the coronary vessels for the complete cardiac cycle. Combined with the measurement of intracoronary pressure changes, the use of IVUS could allow more accurate calculation of elasticity parameters. In fact, this tool has already been used to measure pulmonary artery elasticity in patients with pulmonary hypertension [13].

This study sought to investigate the value of RBP4 in assessing coronary artery elasticity in patients with CHD combined with T2DM, to investigate new therapeutic strategies to improve the prognosis of these patients.

T2DM has been found to influence and participate in the development of CHD. Its effect on vascular endothelial cells and vascular elasticity is particularly significant and has important pathological significance. Endothelial dysfunction is one of the pathological bases of CHD [18]. Quagliaro et al. [19] found that the concentration of superoxide anion radical (O2${}^{-}$) increases during periods of hyperglycemia. This results in oxidative damage, enhances the expression of cysteinyl aspartate specific proteinase-3 (caspase-3), inhibits the expression of B-cell lymphoma-2 (Bcl-2), which has anti-apoptotic effects, and thus induces endothelial cell apoptosis. Another study found that the ratio of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) was reduced in patients with T2DM [20]. In addition, BH2 reduces the bioavailability of BH4 by competitively binding to endothelial-type nitric oxide synthase (eNOS), a key cofactor of eNOS. This action reduces endothelial-derived NO production, which is a key factor in the regulation of vascular tone and endothelial function. Furthermore, Yoshida et al. [21] found that hyperglycemia-induced accumulation of advanced glycated end products (AGEs) in the vessel wall or glycosylation of the vascular extracellular matrix significantly reduces vascular compliance and increases the incidence of CHD in patients with T2DM.

This study found that in addition to T2DM; RBP4, age, hypertension and LDL-C significantly affected coronary artery elasticity, which is generally consistent with the results of previous studies [22, 23, 24, 25]. A study by Chondrou et al. [22] regarding the effect of RBP4 on arterial elasticity, found that RBP4 was significantly associated with aortic stiffness after adjusting for the influence of age and pulse pressure, as RBP4 levels increased, arterial elasticity gradually decreased. Aging is also a known factor contributing to the decrease of the elastic function of large arteries. The effect of age on arterial elasticity is partly attributed to changes in the extracellular matrix within the arterial wall, such as degradation of elastin fibers and increased formation of cross-linked molecules such as AGEs [23]. In addition, ageing-induced imbalance in the vasoactive molecular environment, vascular oxidative stress and chronic inflammation are also thought to reduce arterial compliance. The negative effects of hypertension on vascular elastic function have also been demonstrated in clinical practice. Sustained high circulatory load in patients with hypertension will damage the elastin structure of the arterial wall, causing remodeling of the vessel wall and lumen enlargement, as well as inducing phenotypic changes in arterial endothelial and smooth muscle cells to decrease arterial elasticity [24]. Finally, Chen et al. [25] found in a cross-sectional study, that the risk of atherosclerosis increased with increasing duration of LDL-C exposure in a dose-dependent manner, demonstrating that elevated LDL-C levels will further decrease arterial compliance.

In this study, in order to determine whether the effect of RBP4 on coronary artery elasticity was different in patients with CHD with and without T2DM, a multiple linear regression analysis was established by using the $\beta$ value as the dependent variable in both groups. The results showed that RBP4 could significantly affect the coronary artery elasticity of patients in the CHD with T2DM group but not affect that of patients in the CHD without T2DM group. The potential mechanism of the effect of RBP4 on coronary artery elasticity will require further investigation.

The effect of RBP4 on arterial elasticity may be partly attributed to its induction of insulin resistance in endothelial cells and the reduction of endothelium-derived NO production [26]. The mechanisms of insulin action on arterial endothelial cells have now been extensively elucidated [27, 28]. After binding to endothelial cell surface receptors, insulin can increase endothelial-derived NO production through the PI3K/Akt signaling pathway, and act on the mitogen-activated protein kinase (MAPK) signaling pathway to promote the secretion of endothelin-1 (ET-1), which has a strong vasoconstrictive effect and promotes the expression of adhesion factors. Insulin resistance (IR) could selectively inhibit the PI3K/Akt-eNOS-NO signaling pathway without reducing ET-1 secretion, and the imbalance between the two would impair arterial vasodilatory function [28].

RBP4 can also reduce arterial compliance and accelerate atherosclerosis progression by impairing mitochondrial function and inducing endothelial apoptosis in arterial endothelial cells. The PI3K/Akt signaling pathway not only is involved in regulating endothelial-derived NO production, but also functions to regulate the activity of the Bcl-2 family proteins [29]. An increase in the Bax/Bcl-2 ratio leads to a change in mitochondrial membrane permeability, increasing the release of mitochondrial cytochrome C (Cyt C) and promoting apoptotic events, whereas a decrease in the Bax/Bcl-2 ratio has the opposite effect. Wang et al. [10] found that RBP4 increased mitochondrial reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs) in a dose-dependent manner, and decreased mitochondrial content, integrity and membrane potential. In either RBP4-treated HAECs or the transgenic mice expressing human RBP4 (RBP4-Tg), Cyt C expression and Bax/Bcl-2 ratio were found to be significantly elevated, which would eventually lead to apoptotic events in arterial endothelial cells. Combined with changes in phosphorylation levels at specific downstream sites (Ser473, Thr308), this suggests that high levels of RBP4 will inhibit the PI3K/Akt signaling pathway and induce apoptosis in aortic endothelial cells.

RBP4 may affect coronary artery elasticity by promoting the proliferation and migration of vascular smooth muscle cells (VSMCs), which exhibit a fully functional and differentiated phenotype under physiological conditions and express contractile proteins important for maintaining vascular tone. However, even though VSMCs are highly differentiated and mature, they are still highly plastic and can undergo a phenotypic transition from “contractile” to “synthetic” phenotype under conditions of injury or induction [30]. The phenotypic transition from a “contractile” to a “synthetic” phenotype is characterized by a decrease in myofilament density and contractile protein expression, which are replaced by an increase in the expression of proinflammatory factors and extracellular matrix [31]. For example, VSMCs can be involved in vascular calcification by transforming into osteoblasts. Zhou et al. [32, 33] found that RBP4 promoted the development of atherosclerosis in both diabetic rats by regulating the JAK2/STAT3 signaling pathway and rat aortic smooth muscle cells in a high-glucose environment, further validating that high levels of RBP4 promoted the proliferation of VSMCs by regulating this pathway. This further verified that high levels of RBP4 promoted the proliferation and migration of VSMCs and the occurrence of diabetic macrovascular events through the regulation of this pathway, consistent with the view in our study that RBP4 is a risk factor for the progression of coronary artery lesions in CHD patients with T2DM.

Finally, The relationship between pericardial fat thickness and coronary heart disease has been closely studied in recent years. Epicardial adipose tissue (EAT) volume (EAV) can be used to diagnose high-risk coronary plaque burden associated with coronary events [34]. Further research shows that pericoronary adipose tissue is closely related to atherosclerotic plaque formation. Right coronary artery Pericoronary adipose tissue computed tomography attenuation(PCATa) has prognostic value beyond clinical characteristics [35]. Several studies have found that EAV has a negative relationship with artery stiffness [36, 37]. The mechanism by which coronary peripheral fat or pericardial peripheral fat increases the risk of coronary atherosclerosis remains unclear. Salgado-Somoza et al. [38] showed that Retinol-binding protein 4 is expressed in EAT and subcutaneous adipose tissue (SAT), and that RBP4 protein levels were higher in EAT from CAD than non-CAD patients. Therefore, RBP4 produced by EAT is another risk factor for the progression of coronary artery lesions in CHD. Since RBP4 is produced by adipose tissue, one of the reasons for the increase of RBP4 in CHD patients may be due to the high proportion of adipose tissue or BMI in these patients. Controlling body fat to reduce the level of RBP4 may be an important measure to reduce the occurrence of coronary heart disease in diabetic patients.

Our patients all had diabetes and coronary heart disease and received medication to control blood sugar, hypertension and lipid levels. Weather these medical therapies can influence RBP4 levels is still unclear. These medications and dietary modifications will impact levels of oxidative stress and lifestyle changes such as cessation of smoking could modulate RBP4 concentrations [39]. Fortunately, both our study groups had similar clinical characteristics and these factors had limited impact on our results. In CHD patients, plaque component and plaque elasticity had a significant positive relationship with artery stiffness [40, 41]. Therefore, the left main coronary artery and the proximal part of the right coronary artery without plaque were selected as the locations for measuring arterial elasticity.

Our study is innovative in that the obtained coronary elasticity parameters were analyzed in correlation with RBP4 by using IVU. Furthermore, this study reveals that the adipokine RBP4 is an independent risk factor for coronary artery elasticity and shows differences in levels between CHD patients with and without T2DM, providing a new therapeutic strategy for patients with CHD combined with T2DM. However, this study still has some limitations. First, this study was a cross-sectional study and did not demonstrate the time-dependent effect of RBP4 on coronary artery elastic function. Second, the small sample size was small and derived from a single-center in Suzhou. Third, this study was based on the population with stable CHD, so the results cannot be extended to acute patients. Fourth, our study did not show RBP4 was an independent risk factor for vascular stiffness in patients with CHD without T2DM. It could be that the number of patients without diabetes and with significant vascular stiffness was too low. Finally, the correlation between RBP4, arterial stiffness and clinical outcomes had not been investigated, so further clinical studies are needed to understand whether reducing RBP4 levels in diabetic patients with CHD may actually have an impact on prognosis. This topic will need further investigation future studies.

Our study found that RBP4 was an independent risk factor for coronary artery elasticity in patients with CHD combined with T2DM and in all CHD patients, but it did not affect the coronary artery elasticity of CHD patients without T2DM. This suggests that RBP4 is important for the assessment of coronary artery elasticity in patients with CHD combined with T2DM and that treatment targeting RBP4 may decelerate the progression of coronary artery lesions in these patients.

$\beta$, stiffness parameter; Ep, pressure-strain elastic modulus; DC, distensibility coefficient; CC, compliance coefficient; AGEs, advanced glycated end products; EEM, external elastic membrane; caspase-3, cysteinyl aspartate specific proteinase-3; Bcl-2, B-cell lymphoma-2; BH2, dihydrobiopterin; BH4, tetrahydrobiopterin; eNOS, endothelial nitric oxide synthases; MAPK, mitogen-activated protein kinase; ET-1, endothelin-1; HAECs, human aortic endothelial cells; Bax, BCL2-associated X protein; VSMCs, vascular smooth muscle cells.

The data and materials that support the findings of this study are openly available from the corresponding author upon reasonable resquest.

XG designed the research study. YJ, SD, CT and JS performed the research and analyzed the data. YJ wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University (No. JD-LK-2021-029-01).

This study was funded by the Department of Cardiology of the Second Affiliated Hospital of Soochow University. We thank all the staff for their help with data collection.

This work was supported by the Project of State Key Laboratory of Radiation Medicine and Protection, Soochow University (NO. GZK1202135). Academic lifts project of the Second Affiliated Hospital of Soochow University (NO. XKTJ-HRC2021007).

The authors declare no conflict of interest.