Angiotensin I and angiotensin II concentrations and their ratio in catecholamine-resistant vasodilatory shock

Analyses of baseline ANG I, ANG II, and ANG I/II ratio and association with survival were prespecified. All other analyses, including comparison to healthy controls, were post hoc. Wilcoxon rank-sum test, Fisher’s exact test for binary outcomes, and chi-square statistic for other categorical outcomes were used for comparisons. Survival from the time of randomization to time of death from any cause was analyzed by the Kaplan–Meier formula. Estimates and confidence intervals were calculated by the product limit method and Greenwood’s formula for the variance and included the difference between treatment arms. For missing data in time-to-event analyses, including mortality at day 28, censored data techniques were utilized. Patients with missing data were censored on the last known survival date up to the specified endpoint (i.e., day 28).

The median ANG I/II ratio across treatment arms at baseline was 1.63 (IQR 0.98–5.25). Patient demographics and disease characteristics by baseline median ANG I/II ratio were largely similar between groups (Table 2). However, recent exposure to ACE inhibitors was significantly more common in patients with a ratio above the median. Moreover, patients with a higher ANG I/II ratio had higher serum albumin concentrations and were receiving a higher dose of vasopressor support (norepinephrine-equivalent dose) at baseline. Baseline ANG I/II ratios were similar between the placebo (n = 139) and ANG II treatment arms (n = 142) (Table 2).

Mortality in the trial’s placebo treatment arm was 64.7% in those with baseline ANG I/II ratio above the median and 45.2% in those with a ratio below the median (Fig. 1). In a multivariate analysis of mortality in the placebo arm, the baseline ANG I/II ratio was a significant predictor of overall mortality (hazard ratio 0.54; P = 0.0111) (Table 3, Fig. 1)

Previous studies have reported that the baseline ANG I/II ratio averaged 0.38 in otherwise healthy patients with hypertension [15]; this is consistent with the ratio of 0.4 in healthy sera measured. The median ratio value of 1.63 for patients in the present study suggests a possible pathological decrease in conversion of ANG I to ANG II in patients with CRVS. Endothelial injury is common during septic shock. Thus, endothelial membrane–bound ACE activity may be reduced during shock. Logically, reduced ACE activity should lead to decreased ANG I to ANG II conversion and an increased ratio. A significant proportion of ATHOS-3 patients had high ANG I/II ratios, suggesting decreased ACE activity. Low levels of ANG II and ACE activity on day 1 have been previously reported in patients with sepsis and appear associated with a poor prognosis [17]. Decreased ACE activity could be due to an intrinsic defect in ACE function [20] or to small peptides with ACE inhibitory properties [24]. In addition, at least two pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-1β) downregulate ACE in cultured human endothelial cells [25]. Finally, while not examined in this study, different single-nucleotide polymorphisms of ACE can affect ACE activity and are associated with mortality rates in septic shock [26], possibly through interactions between TNF-α and such polymorphisms [27]. It appears biologically plausible that a high ANG I/II ratio may reflect decreased ACE activity. In keeping with this notion, the recent use of ACE inhibitors was markedly more common in patients with a high ANG I/II ratio in our study.

Another key enzyme, ACE2, can also affect the ANG I/II ratio. ACE2 catalyzes the conversion of ANG II to ANG (1–7) [28], and increased ACE2 activity may also decrease ANG II levels and increase ANG I/II ratios. Therefore, high ACE2 activity may contribute to a high ANG I/II ratio in vasodilatory shock.

Our findings suggest that in many patients with CRVS, there is an imbalance between ANG I and ANG II levels. This imbalance may be related to changes in ACE1 and/or ACE2 activity, which may relatively diminish ANG II generation and can be exacerbated by recent ACE inhibitor administration. Moreover, the findings imply that diminished ability to convert ANG I to ANG II may contribute to a catecholamine-resistant vasodilatory state and increase the risk of death. In their aggregate, these findings suggest that there is a biological rationale for the exogenous administration of ANG II in CRVS.

To our knowledge, this is one of the first studies to evaluate serum ANG I and ANG II levels and the ANG I/II ratio in patients with CRVS. Only a single recent pilot study found that increased ANG I levels were correlated with mortality [29]. In comparison, our study was much larger and involved several hundred patients in multiple countries and continents, thus providing a high level of external validity. In addition, this study utilized a double-blind, placebo-controlled, phase 3 registration trial design, assuring that characteristics and outcomes were collected prospectively and were independently monitored; this minimized selection and ascertainment bias. The measurements of ANG I and ANG II were performed by an independent laboratory blinded to clinical characteristics, thus further minimizing bias. Moreover, the analysis of such data followed a prespecified protocol. Finally, the associations observed appear logical and consistent with current knowledge of the physiology and pathophysiology of ANG I, ANG II, and ACE1 and ACE2 activity in inflammatory states.

Our study had limitations. We dichotomized ANG I/II ratios as part of our assessment. Such an approach simplifies comparisons but is insensitive to the continuous nature of biological variables. Thus, the correct specific cutoff point to inform clinical decisions remains unknown. Follow-up was to 28 days only, so implications for longer survival windows could not be made. In addition, ACE activity was not measured directly; rather, ACE activity was inferred from the ratio of ANG I/II in this study. However, patients with prior exposure to ACE inhibitors appeared to be particularly prone to a high baseline ANG I/II ratio, indicating that, in at least some patients, a high baseline ratio very likely resulted from decreased ACE activity. We did not measure the ANG I/II ratio in real time. However, ANG I and II concentrations were collected prospectively as part of a prespecified analysis. We did not measure ACE2 activity as part of the ATHOS-3 study. Thus, our suggestion that increased ACE2 activity may affect the ANG I/II ratio remains speculative. Further studies will require a more detailed assessment of the increasingly complex angiotensin family of molecules and their interaction with ACE1 and ACE2 activity.