Severe endothelial injury and subsequent repair in patients after successful cardiopulmonary resuscitation

The clinical course of patients after successful cardiopulmonary resuscitation (CPR) is often complicated by post-resuscitation disease, a condition of multiple life-threatening disorders related to whole-body ischemia and reperfusion syndrome [1, 2]. This phenomenon shares many features with severe sepsis, including a systemic inflammatory response with plasma cytokine elevation, coagulation abnormalities, and myocardial dysfunction [3]. Ischemia, reperfusion and hypoxia during or after CPR induce generalized tissue damage with release of reactive oxygen species and endothelial-leukocyte interaction and activation, resulting in increased microvascular permeability and, hence, in loss of endothelial integrity [2]. Several studies demonstrated an endothelial activation with a consecutive endothelial injury following cardiac arrest [4‐6] and in models of ischemia and reperfusion [7, 8].

A new tool for evaluation of endothelial injury is detection of circulating endothelial cells (CECs): these cells detach from the intima monolayer in response to endothelial damage and become measurable in peripheral blood. Although CECs are rarely found in the blood of healthy individuals, raised numbers are present in patients with a wide variety of diseases involving the endothelium such as vasculitis [9], arterial occlusive disease [10], and cardiovascular disease [11]. There is a strong relation between CECs and endothelial dysfunction because numbers of CECs correlate with surrogate markers of disturbed endothelial function such as flow-mediated dilation and von Willebrand factor (vWF) [12]. Plasma vWF is one of the most established plasma surrogate markers of endothelial damage or dysfunction [13]. VWF antigen concentrations are elevated on the second day after CPR and seem to be an early predictor of outcome [14].

Additionally, there is an increase of endothelial microparticles (EMPs) in states of disturbed endothelial function. Microparticles are small shed membranous vesicles (<1 μm) that are released from cells upon activation or during apoptosis. They reflect the state of their parental cells in amounts and phenotypes [15]. Microparticles have procoagulant properties, modulate endothelial function, and play a role in inflammatory processes [16]. EMPs were found to be elevated in peripheral blood of patients suffering from acute coronary syndrome [17], or peripheral arterial disease [18]. Circulating endothelial progenitor cells (EPCs) are capable of repairing damaged endothelium and furthermore contribute to re-endothelialization and neovascularization [19]. These cells are bone marrow-derived pluripotent vascular progenitor cells that home in on the sites of ischemia and vascular injury [20]. A decrease in EPC count in peripheral blood is associated with endothelial dysfunction [21]. Patients with coronary artery disease showed reduced levels of EPCs [22] and there was an inverse correlation of number of EPCs in the peripheral blood, increased atherosclerotic risk factors, and a higher cardiovascular morbidity and mortality [23].

In the present study, we hypothesize that endothelial injury takes place during and after CPR, which in turn may contribute to post-resuscitation disease. Therefore, the aim of the present study is to detect direct markers of endothelial damage such as CECs, EMPs and vWF, as well as markers of endothelial repair (EPCs) in peripheral blood of patients after successful CPR.

The vascular endothelium lining the blood vessels is one of the most complex and sensible organ systems. Its integrity is an important precondition for a regular function of the circulatory system. The endothelium governs a host of fundamental physiologic functions: endothelial cells serve as signal transduction cells for a variety of chemical and mechanical stimuli, produce cytokines, or other paracrine signalling molecules, regulate leukocyte adhesion and migration, and maintain the balance between pro- and anticoagulant mechanisms [25, 26].

The data presented in this report show the occurrence of severe endothelial damage in patients after successful CPR, and in a follow-up study, of a subsequent endothelial regeneration. In summary, we found a rapid and severe increase in CECs directly after CPR and a delayed increase in EMPs. This was paralleled by elevated levels of vWF, immediately and 24 hours after ROSC, compared with normal values. We chose vWF to support the flow cytometric measurements by a conventional and well-established marker of disturbed endothelium [13]. As levels of vWF showed a positive correlation with CEC count in resuscitated patients, these data underline the reliability of CEC count in the detection of endothelial damage. These results indicate early endothelial damage and ongoing endothelial dysfunction detected by elevated CECs and EMPs in resuscitated patients compared with control groups. Furthermore, numbers of EPCs increased on the second day after ROSC, which points to an early initiation of endothelial regeneration. The direct comparison with patients with stable CAD undergoing coronary intervention excluded effects possibly caused by CAD or coronary intervention.

In this study we could also demonstrate a significant positive correlation between the CEC count and the duration of CPR. In the literature, a long period of ischemia and longer duration of resuscitation efforts are associated with poor outcome [27, 28], and the timing of the ischemic insult and the length of the reperfusion have been shown to correlate with endothelial dysfunction variables and biochemical or histological evidence for cellular damage [25, 29]. Therefore, the correlation described in this study might reflect a greater extent of endothelial damage occurring during longer periods of CPR. However, we have to state that this positive correlation is largely due to a small number of patients with a long duration of CPR with CEC values outside the data cloud.

After cardiac arrest and mechanical resuscitation, there is growing evidence for an ischemia and reperfusion syndrome resulting in several inflammation cascades, including activation of leucocytes [30], up-regulation of selectins [4, 6], and adhesion molecules [5, 31] on the surface of the endothelium. Adrie and colleagues reported a 'sepsis-like syndrome' after resuscitation, with an increase in circulating interleukins, TNF, and a generalized systemic inflammatory response [3]. Researchers recently stated that an early toll-like receptor 4-induced vascular injury might be an important trigger of the systemic inflammatory response in resuscitated mice [32]. In analogy to the data obtained in our study, there are reports of elevated numbers of CECs in critically ill patients presenting with acute respiratory distress syndrome [33] and severe inflammatory disease conditions such as septic shock [34]. Hence, we excluded patients with septic shock from the study. The relatively few patients in the study with in-hospital arrests and the high percentage of patients with cardiac-related arrest suggest that most patients were not critically ill prior to cardiac arrest. Moreover, in critically ill patients, who did not undergo CPR, CEC counts were only slightly elevated (comparable with the levels of the CAD group; data not shown). Therefore, it seems to be unlikely that critical illness or ICU procedures themselves contribute to the elevation of CECs. The currently available data does not allow for a clear differentiation of whether the increase of CECs is the cause or effect of the inflammatory reaction taking place. The very early elevation of CECs to a vast extent after CPR might be an indicator that endothelial damage could be the initial step in post-resuscitation disease resulting in secondary inflammatory reactions. Conceivable triggers for endothelial damage might be hypoxia [30] or shear stress [35, 36]. Therefore, it seems possible that shear stress during mechanical chest compression or the action itself is another cause of enhanced detachment of CEC from the endothelial layer.

We obtained comparatively high absolute values of CECs in this study, compared with the literature. In our opinion this indicates massive endothelial damage after CPR that largely exceeds the values detected in other diseases described so far in the available literature. This underlines the severity of this life-threatening condition, associated with complete discontinuation of circulation and high mortality. On the other hand, CEC counts in the literature vary from 15 to 670 cells/mL in various disease states [33, 37], which points out the inhomogeneity of this relatively new method. Furthermore, different methodical approaches might change the absolute values.

Interestingly, and in contrast to the CECs, the number of EMPs in resuscitated patients rises further in the first 24 hours after ROSC, hence reflecting an ongoing process of endothelial damage. As EMPs are elevated in several systemic inflammatory diseases such as vasculitis [38] and sepsis [39], the noticeable increase in EMP numbers could be due to the systemic inflammatory response occurring after CPR maintaining endothelial injury. EMP may express adhesion molecules specific to mature endothelial cells, such as platelet-endothelial cell adhesion molecule-1 (CD31), VE-cadherin (CD144), or MCAM (CD146). Activation of endothelial cells with TNF-α induced the formation of EMPs [16] exposing adhesion-cell molecules, including E-selectin (CD62E) or intercellular adhesion molecule-1 (CD54). In this study, we measured activation-induced EMPs by detection of E-selectin-positive microparticles. A possible explanation for the ongoing endothelial injury in the post-resuscitation period could be ischemia and reperfusion during cardiac arrest and mechanical resuscitation.

Interestingly, patients treated with statins prior to cardiac arrest showed slightly lower EMP counts. These results indicate a potential protective effect of statins on the endothelium during and after ischemia and reperfusion and encourage further investigation of the effect of statin treatment in post-resuscitation care.

Finally, in a smaller population, we were able to detect elevated numbers of EPC in patients on the second day after CPR as an indicator of the early onset of endothelial repair. EPC-mediated vascular repair has been shown to be associated with normalization of endothelial function and restoration of blood flow at the site of injury [21]. These circulating cells are capable of endothelial differentiation and homing to ischemic tissues [20]. EPCs are considered to originate from hematopoietic stem cells, which are positive for CD34 and VEGF-R2 and immature marker protein CD133 [40]. CD34+ blood cell counts are widely used to obtain 'mobilized' hematopoietic stem/progenitor cells from peripheral blood [41]. Catecholamines are known to induce angiogenesis in tumor tissues [42] and dopamine has been shown to mobilize EPCs from the bone marrow during tumor growth [43]. Nevertheless, norepinephrine failed to increase CD34+ levels in heart failure patients [44]. In the EPC study group, all CPR patients received vasopressors such as norepinephrine and epinephrine but none of them received dopamine. Moreover, a small control group of critically ill patients at our ICU, who did not undergo CPR but received vasopressors and mechanical ventilation, showed no elevation of EPCs (data not shown). However, we cannot exclude the possibility of any influence of catecholamines or ICU procedures administered.

A limitation of the present study is its observatory design. Further experimental studies could allow a more detailed investigation of causes and effects of endothelial damage in post-resuscitation disease. Moreover, experimental studies would allow the assessment of possible therapeutic interventions. In the current study, the number of patients was rather small, particularly in the EPC study. However, the described effects are overwhelming and obtain clear statistical significance.

In this study we provide evidence for an endothelial injury occurring in patients after CPR. The obtained data suggest a two-step process: The early stage during and directly after CPR is prevailed by severe endothelial damage. Within the following 24 hours, inflammation and endothelial repair are taking place. These results could be the basis for further interventional studies with the aim of developing new therapeutic and prognostic strategies in post-resuscitation care. As a perspective, endothelial protection (e.g. statins), anti-inflammatory drugs (e.g. corticosteroids), as well as regenerative therapies (e.g. growth factors) could be promising future therapeutic strategies in the early phases after CPR.