The association of interferon‐alpha with development of collateral circulation after artery occlusion

Abstract Background Previous studies have demonstrated that interferon (IFN) signaling is enhanced in patients with poor collateral circulation (CC). However, the role and mechanisms of IFN‐alpha in the development of CC remain unknown. Methods We studied the serum levels of IFN‐alpha and coronary CC in a case–control study using logistics regression, including 114 coronary chronic total occlusion (CTO) patients with good coronary CC and 94 CTO patients with poor coronary CC. Restricted cubic splines was used to flexibly model the association of the levels of IFN‐alpha with the incidence of good CC perfusion restoration after systemic treatment with IFN‐alpha was assessed in a mice hind‐limb ischemia model. Results Compared with the first IFN‐alpha tertile, the risk of poor CC was higher in the third IFN‐alpha tertile (OR: 4.79, 95% CI: 2.22–10.4, p < .001). A cubic spline‐smoothing curve showed that the risk of poor CC increased with increasing levels of serum IFN‐alpha. IFN‐alpha inhibited the development of CC in a hindlimb ischemia model. Arterioles of CC in the IFN‐alpha group were smaller in diameter than in the control group. Conclusion Patients with CTO and with poor CC have higher serum levels of IFN‐alpha than CTO patients with good CC. IFN‐alpha might impair the development of CC after artery occlusion.


| BACKGROUND
Development of collateral circulation (CC), also termed as arteriogenesis, is a natural life-preservation mechanism occurring in patients with coronary chronic total occlusion (CTO). Well-developed CC preserves cardiac function, thus reducing cardiac mortality after coronary occlusion. 1,2 Patients with CTO show a great deal of heterogeneity in their arteriogenic responses to artery occlusion. This is affected by multiple factors, including inflammatory factors. 3 The interaction between inflammation and arteriogenesis may be worthy of study, but data on the correlation between arteriogenesis and inflammation are limited. A previous study has found that interferon (IFN) signaling in monocytes is enhanced and stimulated by lipopolysaccharide among patients with impaired coronary CC. 4 Furthermore, blocking the receptor of IFN have been found to be helpful in alleviating ischemia in mice. 5 However, whether patients with poor CC have higher levels of serum IFN or whether treatment with IFN inhibits the development of arteriogenesis remains unknown. IFN-alpha is widely used in treating hepatitis B and C, as well as various cancers.
IFN-alpha might affect the development of CC in patients with CTO.
In this study, we aimed to investigate whether patients with poor CC have higher levels of IFN-alpha, and if so, whether IFN-alpha inhibits the development of CC.

| Study population
This study was conducted in accordance with the Declaration of Helsinki and with written informed consent of each participant. This study was approved by the medical ethics committee of Second Xiangya Hospital of Central South University. Patients undergoing coronary angiography at our catheter laboratory between January 2018 and June 2019 and with at least one major coronary artery total occlusion were enrolled in our study. The exclusion criteria were as follows: (1)  were blinded to the characteristics of the included patients. They reviewed the angiography results and classified the extent of coronary circulation by the Rentrop classification from 0 to 3 as follows: 0 = none, 1 = filling of side branches of the artery dilated via collateral channels without visualization of the epicardial segment, 2 = partial filling of the epicardial segment via collateral channels, and 3 = complete filling of the epicardial segment of the artery being dilated via collateral channels. 6 Rentrop 0-1 were classified as poor CC development; Rentrop 2-3 were classified as good CC development. Venous blood samples were collected immediately before coronary angiography. Serum was separated at 4 C, and the levels of IFN-alpha were measured by ELISA (BMS216, Invitrogen).

| Ischemic hind-limb model and laser speckle imaging
The animal protocol was approved by the animal ethics committee of University. Male C57/BL mice were anesthetized with 3% isoflurane.
The left femoral artery was ligated and excised as described previously. 7 The right leg underwent sham operation and was used as control. The mice were randomly divided into two groups: an INF-alpha group with intraperitoneal injection of 2000 IU IFN-alpha, and a control group with intraperitoneal injection of the same amount of saline.
The IFN-alpha concentration used is comparable to dosages used in patients. Hind-limb prefusion was measured by laser speckle imaging under temperature-controlled conditions, before and after left hindlimb ligation, as well as at 3 days, 1 week, and 3 weeks following ligation. Color-coded images of the paws representing the flux value were used to calculate the ratio of the tissue perfusion of the occluded (left) to the Sham-operated (Sham) paw. The right hind-limb was used as reference. All mice were put to death by cervical dislocation.

| Histological analyses
Adductor muscles from bilateral hind limbs were harvested at 3 weeks after surgery and underwent immediate tissue fixation overnight. For mouse arteriole density identification, the adductor muscles were stained with alpha-SMA monoclonal antibody at 3 weeks after ligation.

| Statistical analyses
We presented baseline characteristics of patients as frequencies and percentages for categorical variables and as means and standard deviations or interquartile range for continuous variables, depending on whether data distribution was normal (assessed by normal Q-Q plots).
We compared categorical variables using chi-square analysis, and con-  We also did several sensitivity analyses by excluding patients with age <55 years or >70 years or by using the quantiles for the levels of serum IFN-alpha. We performed all of the analyses using R version 3.4.3 (R Foundation for Statistical Computing, Vienna, Austria).

| RESULTS
Between January 2018 and June 2019, 208 patients with CTO were included in our study as required by the inclusion and exclusion criteria ( Figure S1). The baseline characteristics of the included patients are presented in Table 1 Table 2). After adjusting for model 3, the cubic spline-smoothing curve shows that the risk of poor CC increased with increasing serum IFN-alpha levels ( Figure 2 Note: When the levels of IFN-alpha larger than 112 pg/mL, the incidence of poor CC did not increase with the increase of the levels of IFN-alpha. (59.8% ± 3.31% vs. 66.5% ± 3.21%, p = .005) and at 21 days (59.8% ± 3.31% vs. 66.5% ± 3.21%, p = .005, Figure 3A,B). Although the numbers of arterioles are comparable between groups, the diameter of the arterioles in the mice treated with IFN-alpha was smaller compared with the control group ( Figure 4A). In order to study local ischemia, we examined the morphology in the gastrocnemius muscle by HE staining and Masson staining. Although we did not find obvious morphological changes by HE analysis (Figure S2), the area of interstitial fibrosis evaluated by Masson staining in the mice treated with IFN-alpha was larger than in the control group ( Figure 4B).

| DISCUSSION
In this study, we first found that CTO patients with poor CC have higher serum levels of IFN-alpha. We found that IFN-alpha inhibits the development of CC after artery occlusion.

CONFLICTS OF INTEREST
The authors declare that they have no potential conflicts of interests.

AUTHOR CONTRIBUTIONS
Xinqun Hu and Zhenhua Xing designed the study and provided methodological expertise. Zhenhua Xing drafted the manuscript. Zhenhua Xing performed the case-control study. Xiaopu Wang, Junyu Pei, Zhaowei Zhu, Shi Tai performed the animal experiments. All authors have read, provided critical feedback on, and approved the final manuscript.

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.