Sequential Surgical Procedures in Vascular Surgery Patients Are Associated With Perioperative Adverse Cardiac Events

Abstract

Patients at elevated cardiovascular risk are prone to perioperative cardiovascular complications, like myocardial injury after non-cardiac surgery (MINS). We have demonstrated in a mouse model of atherosclerosis that perioperative stress leads to an increase in plaque volume and higher plaque vulnerability. Regulatory T cells (Tregs) play a pivotal role in development and destabilization of atherosclerotic plaques. For this exploratory post-hoc analysis we identified 40 patients recruited into a prospective perioperative biomarker study, who within the inclusion period underwent sequential open vascular surgery. On the basis of protein markers measured in the biomarker study, we evaluated the perioperative inflammatory response in patients' plasma before and after index surgery as well as before and after a second surgical procedure. We also analyzed available immunohistochemistry samples to describe plaque vulnerability in patients who underwent bilateral carotid endarterectomy (CEA) in two subsequent surgical procedures. Finally, we assessed if MINS was associated with sequential surgery. The inflammatory response of both surgeries was characterized by postoperative increases of interleukin-6,-10, Pentraxin 3 and C-reactive protein with no clear-cut difference between the two time points of surgery. Plaques from CEA extracted during the second surgery contained less Tregs, as measured by Foxp3 staining, than plaques from the first intervention. The 2nd surgical procedure was associated with MINS. In conclusion, we provide descriptive evidence that sequential surgical procedures involve repeat inflammation, and we hypothesize that elevated rates of cardiovascular complications after the second procedure could be related to reduced levels of intraplaque Tregs, a finding that deserves confirmatory testing and mechanistic exploration in future populations.

Keywords: cardiac adverse event; interleukin-6; perioperative inflammation; plaque vulnerability; regulatory T cells; vascular surgery.

Figure 1

Patient flow chart. CEA, Carotid endarterectomy.

Figure 2

Perioperative systemic inflammation. Measurements were performed in pre- and postoperative plasma samples of 1st and 2nd surgery. (A) IL-6 measured by CBA, (B) IL-10 measured by CBA, (C) PTX3 measured by ELISA, (D) CRP taken from routinely performed perioperative blood work. Graphs are displayed as median with whiskers extending from 5th to 95th percentile. Data were compared using Kruskal–Wallis test for global assessment of differences in the data family before limited group wise comparisons were done using Dunn's test. To account for multiple comparisons, statistical analyses of plasma were adjusted according to Bonferroni (α <0.05/4). Overall, both surgeries led to a similar inflammatory response associated with detection of markers for increased plaque vulnerability. IL, Interleukin; PTX3, Pentraxin 3; CRP, C-reactive protein.

Figure 3

Histological assessment of atherosclerotic lesions from carotid endarterectomy. CEA samples of 17 patients who underwent bilateral CEA were selected for histological assessment. (A,B) Representative H&E staining of CEA samples from the same patient of 1st and 2nd surgery. (C) Relative necrotic core size did not differ between 1st and 2nd CEA sample. (D,E) Representative immune fluorescent staining of Collagen I of CEA samples from the same patient of 1st and 2nd surgery (F) Relative Collagen I content did not differ. (G,H) Representative immune fluorescent staining of αSMA of CEA samples from the same patient of 1st and 2nd surgery. (I,J) Relative content of αSMA and minimal thickness of fibrous cap did not differ. (K) Virmani Classification of samples harvested at both surgeries. Overall, plaques were more advanced in the 1st surgical cohort. (C,F,I,J) Wilcoxon matched-pairs signed rank test. Data is presented as median with whiskers extending from 5th to 95th percentile. (K) Chi-square test. H&E, Hematoxylin & Eosin; L, Lumen, NC, Necrotic core; FC, Fibrous cap; αSMA, alpha-smooth muscle actin.

Figure 4

Assessment of plaque vulnerability based on leukocyte subpopulation content. (A,B) Representative immune fluorescent staining of CD68 of CEA samples from the same patient of 1st and 2nd CEA. (C) Relative content of CD68 did not differ between 1st and 2nd CEA sample. (D) H&E staining of the sample from the 1st CEA was used for identification of plaque shoulders. (E) Representative staining of CD4 and (F) of FOXP3 on a patient's sample from the 1st CEA. Arrows indicate CD4 and FOXP3 positive cells, respectively. (G) H&E staining of the same patient on the sample from the 2nd CEA. (H) Representative staining of CD4 and (I) FOXP3 on the sample from the 2nd CEA. Arrows indicate positive cells. (J) Content of CD4 positive cells did not differ between 1st and 2nd CEA sample in areas identified as plaque shoulders. (K) Samples from the 2nd surgery contained less FOXP3 positive cells in plaque shoulders. (C,J,K) Wilcoxon matched-pairs signed rank test. Data is presented as median with whiskers extending from 5th to 95th percentile. L, Lumen; H&E, Hematoxylin & Eosin; NC, Necrotic core; FC, Fibrous cap.

Figure 5

Incidence of myocardial injury after first and second surgical procedure. (A) After the 1st surgery one patient was diagnosed with MINS, after the 2nd surgery seven patients suffered MINS. The 2nd surgical procedure was associated with MINS (OR 8.1, 95% CI [1.3; 93.3]). (B) In the subgroup of patients undergoing consecutive CEA, no patient suffered MINS after the first procedure and three after the second. (C) Display of patients with any other combination of surgery. One patient was diagnosed with MINS after the first procedure and four after the second. Chi-square test. Data are presented as absolute numbers per group. CEA, Carotid endarterectomy.