**1. Introduction**

Chromogranins proteins had attracted the interest of scientists and clinicians since the mid-sixties when Blaschko H et al. [1] and Helle KB [2] released the first two manuscripts stored in the PubMed database on this topic. Since then, no less than 9891 papers have been published on chromogranins, of which, however, only 19 are indicated as concerning data recorded in patients admitted in critical care wards (Figure 1). The interest in chromogranins as mediators of life-stressing diseases on human health, although thus only at an early stage, has been slowly growing over the last decade. Fifteen years ago, our study

**Citation:** Schneider, F.; Clère-Jehl, R.; Scavello, F.; Lavigne, T.; Corti, A.; Angelone, T.; Haïkel, Y.; Lavalle, P. Chromogranin A and Its Fragments in the Critically Ill: An Expanding Domain of Interest for Better Care. *Pharmaceutics* **2022**, *14*, 2178. https://doi.org/10.3390/ pharmaceutics14102178

Academic Editor: Rakesh Tiwari

Received: 18 August 2022 Accepted: 9 October 2022 Published: 12 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

group was among the first to consider CGA as a possible object of examination in critical illness. Yet, this area of assessment is complicated. First, in vivo studies require testing large cohorts of patients with clinical phenotypes as similar as possible. This necessitates extensive multicenter studies when clinically relevant hypotheses have been validated in feasibility studies. For instance, now that we have identified Vasostatin-I (VS-I) as a myocardial depressant factor [3], it becomes interesting to gather data to comprehend its role in depressed cardiac contractility in acute hypokinetic shock. Second, clinicians must test in vivo clear-cut null hypotheses, which may be very difficult in multiple organ failure patients. As an example of such difficulty, systemic inflammation in response to strong pro-inflammatory triggers is frequent in intensive care units (ICUs), but in vivo studies require the assessment of chromogranins at the same stage of a given disease mandatorily. In each setting, pharmacokinetics, pharmacodynamics, and the volume of distribution of the proteins of interest are not currently established; and this is independent of the availability of adequate techniques of measurements that may vary according to the characteristics of both the test used and the physiological liquids from which samples have been harvested. Finally, having decided to assess the presence of these proteins, adequate biological sampling techniques must be chosen. This sometimes requires the in-house development of biological assays [4].

**Figure 1.** Number of annual papers issued in the PubMed database according to the query from 1967 to 2022. Please note that the scale of measurement is ten times larger for the query "Chromogranin" (left vertical axis) than for the query "chromogranin and critical care" (right vertical axis).

Patients admitted to an ICU for a disease with organ failure are at risk of short-term death. In this setting, in response to the initial assault on their integrity, they develop a systemic inflammatory response to maintain vital organs functioning and optimal tissue repair. This response is temporary and well-balanced between pro- and anti-inflammatory mediators; it results in recovery, provided the initial trigger of the disease does not overwhelm physiological responses. Following initial local injury, the organism develops (i) a

hormonal, metabolic response, (ii) an immunological response with a neurological component, and (iii) a hemodynamic response called "acute circulatory failure" or shock [5,6]. During that period, the organism rapidly releases chromogranins not only from adrenal medulla stockpiles but also from the diffuse neuroendocrine cell islets existing throughout the organism. From blood concentration assessments, physicians can indirectly assess the balance between synthesis, release, and clearance of the molecules while also considering the possible impact of extra-renal purification techniques.

Bearing these precautions in mind, we report our experience on CGA (and related peptides) in critically ill and experimental conditions related to acute and severe illnesses.

### **2. CGA and Vasostatin-I (CGA1-76) Are Biomarkers of Severity in the Critically Ill**

In 2007, we assessed whether CGA could become a biomarker of severity in lifethreatening diseases on hospital admission. Our primary aim was to improve the triage of intermediate-severity patients to select the most adapted care for them. At that time, the agreed practice was to assess severity in ICU patients with the Simplified Acute Physiology Score 2 (SAPS II) based on a European/North American multicenter study [7]. This score provides an accurate estimate of the risk of death in the ICU without having to specify a primary diagnosis. It proved efficient notwithstanding that since it was available only at hour 24 of admission, it could not become a tool for early triage, whereas a rapidly available and performant biological test would.

The idea of assessing CGA was prompted by the fact that life-threatening diseases trigger a physiological response to an aggressive challenge to health with a triple component: (i) a hormonal, metabolic response; (ii) an immunological response, and (iii) a hemodynamic response—all of which are also linked by a neural immunity balance [6]. Following damage to the body's integrity, a patient will activate the release of various hormones, such as adrenalin and cortisol, as well as many others [8]. The adrenocortical response is similar in all mammals: afferent impulses from receptors implanted in the injury site stimulate the secretion of hypothalamic-releasing factors, further stimulating the pituitary gland. Consequently, the adrenal cortex releases cortisol and the adrenal medulla adrenaline together with chromogranins that are stored and co-released by the chromaffin cells. A feasibility study confirmed the hypothesis that patients with acute organ failure present at admission increased serum concentrations of CGA as a probable component of the early hormonal "fight-or-flight" adaptive response to stress [9]. Importantly, circulating CGA concentrations increased in association with systemic inflammation rather than with infection and the SAPS II, suggesting a possible link with either survival or one of the components of the score. These data align with what happens in humans even in the context of a stressing injury less severe than trauma, thus indicating that the slightest injury to the skin integrity also results in a signal of stress with the release of chromogranin-derived anti-microbial peptides (AMP) [10]. In a confirmation study [11], we reported that CGA concentrations correlate better with systemic inflammation (assessed by both C-reactive protein and procalcitonin) than with infection. Yet, CGA levels reached the highest values in septic shock. In addition, admission CGA values were equivalent to the SAPS II in predicting 28-day mortality. This offered the attractive opportunity for assessing outcomes as early as a few hours after admission in patients free of any other cause of CGA increase.

Further insight into the use of CGA as a biomarker of triage required an extension of the study size. We wondered whether a single admission dosage of VS-I (CGA-1-76), the major N-terminal fragment, would enable greater accuracy than a single CGA test. In a pilot prospective observational study performed in third-level French ICUs, we demonstrated that admission concentrations of VS-I were increased when compared with the values recorded in controls, and this was even more so when the shock was present [12]: VS-I concentrations above 3.97 ng/mL were indicative of poor outcome. Furthermore, including arterial lactate and age in the prediction model improved the reliability of the assessment, making it significantly better than the SAPS II as early as 4 h after admission.

In a further study, we investigated whether CGA could be a marker of the severity of the initial challenging disease as regards its impact on morbidity. Care-related infections are a major issue in critical care for many reasons (costs, bed- blocking and emergence of antibiotics-resistant superbugs). We chose the study phenotype of "multiple trauma" in patients previously in good health. This model appeared more suitable for investigating the impact of a basic injury on health, given that older patients with comorbidities were excluded. The study model, by definition, included a "two-hit" challenge to health: first, the trauma itself and, shortly thereafter, the surgical procedure, which represents a second hit for CGA release. In such patients, we noted an altered plasmatic CGA response revealing a potential mechanism for an association with nosocomial infection [13]. Over several days from admission, CGA concentrations increased compared with control values, but they also leveled off at a relatively high level associated with acute renal failure. Importantly, admission values of CGA significantly increased in those multiple trauma patients who subsequently developed a nosocomial infection: a concentration of 67 ng/mL predicted this occurrence with a sensitivity of 100% and a specificity of 70%, leading to a negative likelihood ratio of almost zero. Recently, we confirmed that admission CGA achieved similar performance for predicting nosocomial infections in COVID patients requiring oxygenation support [14]. This suggests that CGA may reflect not only the triggering disease but also the neuro-hormonal response by the neuroendocrine tissues. In multiple trauma, we finally investigated the ability of VS-I to modulate the innate response of monocytes that are called on to upgrade the patient's defense [13]. Acting as a cell-penetrating peptide, the CGA47-70 fragment, not however including either its scrambled or its prolonged isoforms, was able to downregulate both NF-kappa B and AP-1: this suggests an indirect anti-inflammatory pathway potentially entailing a risk of temporary immune deficiency. These data support the possible occurrence of some forms of care-related infections when proteases of CGA are upregulated.
