Effects of Remote Ischaemic Preconditioning on the Internal Thoracic Artery Nitric Oxide Synthase Isoforms in Patients Undergoing Coronary Artery Bypass Grafting

Remote ischaemic preconditioning (RIPC) is a medical procedure that consists of repeated brief periods of transient ischaemia and reperfusion of distant organs (limbs) with the ability to provide internal organ protection from ischaemia. Even though RIPC has been successfully applied in patients with myocardial infarction during coronary revascularization (surgery/percutaneous angioplasty), the underlying molecular mechanisms are yet to be clarified. Thus, our study aimed to determine the role of nitric oxide synthase (NOS) isoforms in RIPC-induced protection (3 × 5 min of forearm ischaemia with 5 min of reperfusion) of arterial graft in patients undergoing urgent coronary artery bypass grafting (CABG). We examined RIPC effects on specific expression and immunolocalization of three NOS isoforms — endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) in patients’ internal thoracic artery (ITA) used as a graft. We found that the application of RIPC protocol leads to an increased protein expression of eNOS, which was further confirmed with strong eNOS immunopositivity, especially in the endothelium and smooth muscle cells of ITA. The same analysis of two other NOS isoforms, iNOS and nNOS, showed no significant differences between patients undergoing CABG with or without RIPC. Our results demonstrate RIPC-induced upregulation of eNOS in human ITA, pointing to its significance in achieving protective phenotype on a systemic level with important implications for graft patency.

The Effect of Remote Ischaemic Preconditioning on the Immune Response

<p>Remote ischaemic preconditioning (RIPC) describes the phenomenon where brief intermittent periods of limb ischaemia are used to protect the heart and other organs from subsequent prolonged ischaemic insults. RIPC has been identified as a promising intervention for use during cardiac surgery and has consistently shown a beneficial effect in animal models; however, the results of early clinical trials have not been as successful. The exact mechanisms involved in mediating RIPC have not yet been characterised and a better understanding of the pathways through which RIPC exerts its protective effects will be essential in order to progress the translation of this intervention into the clinical setting. There is increasing evidence that RIPC modifies the inflammatory response, therefore the central aim of the research presented in this thesis was to investigate how RIPC affects the human immune system. We performed a double-blind randomised controlled trial of RIPC in 96 high-risk cardiac surgery patients and found no evidence that the intervention reduced myocardial injury or altered peri-operative expression levels of the key inflammatory cytokines, interleukin (IL)-6, IL-8, and IL-10, during simple or more complex procedures. There was a trend towards higher levels of IL-6 and IL-8 in the preconditioned patients; however, confounding variables in the trial design and the heterogeneous patient population limited our ability to interpret the results. We next conducted a paired-analysis trial with 10 healthy male volunteers to assess the direct effect of preconditioning on the early immune response, away from any form of ischaemic injury or comorbidities. We found that RIPC directly and significantly decreased serum levels of the chemokines MIP-1α and MIP-1β, but did not increase the serum concentrations of a range of key cytokines or alter the cytokine producing potential of peripheral blood leukocytes. These findings strongly suggest that a cytokine is not likely to be the humoral mediator associated with transmitting the RIPC protective signal. RIPC did not alter the immunophenotype or extravasation of peripheral leukocyte populations, or the proliferative and cytokine responses of peripheral blood mononuclear cells (PBMC) to pharmacological, physiological, and antigen-specific stimuli. However, preconditioning did appear to reduce the ability of monocytes and neutrophils to respond to activation signals, as indicated by lower levels of CD11b expression in stimulated cultures, and a significant increase in the basal production of IL-22 was also detected in PBMC cultured for 6 days following preconditioning. These alterations may reduce neutrophil and monocyte tissue infiltration and limit the inflammatory response during the early window of RIPC-induced protection and enhance tissue and wound repair several days later. A multivariate analysis confirmed that there was a significant difference in the response between the control and RIPC treatments and the main contributing factors were identified as changes in neutrophil and T cell activation, serum levels of MIP-1α and β, and production of IL-10 and IL-22 from PBMC cultured for 6 days. Overall, our results suggest that RIPC has a subtle but direct effect on the systemic innate immune response during the early window of protection in healthy volunteers, whereas the effects on the adaptive immune system seem to be considerably delayed. The changes detected following RIPC are likely to contribute to protection against ischaemia-reperfusion injury but not solely account for the extent of the beneficial effects of RIPC detected in animals. Our findings reinforce the safety profile of this intervention and have defined a number of immune parameters that are altered by preconditioning for focusing future research.</p>

The Effect of Remote Ischaemic Preconditioning on the Immune Response

<p>Remote ischaemic preconditioning (RIPC) describes the phenomenon where brief intermittent periods of limb ischaemia are used to protect the heart and other organs from subsequent prolonged ischaemic insults. RIPC has been identified as a promising intervention for use during cardiac surgery and has consistently shown a beneficial effect in animal models; however, the results of early clinical trials have not been as successful. The exact mechanisms involved in mediating RIPC have not yet been characterised and a better understanding of the pathways through which RIPC exerts its protective effects will be essential in order to progress the translation of this intervention into the clinical setting. There is increasing evidence that RIPC modifies the inflammatory response, therefore the central aim of the research presented in this thesis was to investigate how RIPC affects the human immune system. We performed a double-blind randomised controlled trial of RIPC in 96 high-risk cardiac surgery patients and found no evidence that the intervention reduced myocardial injury or altered peri-operative expression levels of the key inflammatory cytokines, interleukin (IL)-6, IL-8, and IL-10, during simple or more complex procedures. There was a trend towards higher levels of IL-6 and IL-8 in the preconditioned patients; however, confounding variables in the trial design and the heterogeneous patient population limited our ability to interpret the results. We next conducted a paired-analysis trial with 10 healthy male volunteers to assess the direct effect of preconditioning on the early immune response, away from any form of ischaemic injury or comorbidities. We found that RIPC directly and significantly decreased serum levels of the chemokines MIP-1α and MIP-1β, but did not increase the serum concentrations of a range of key cytokines or alter the cytokine producing potential of peripheral blood leukocytes. These findings strongly suggest that a cytokine is not likely to be the humoral mediator associated with transmitting the RIPC protective signal. RIPC did not alter the immunophenotype or extravasation of peripheral leukocyte populations, or the proliferative and cytokine responses of peripheral blood mononuclear cells (PBMC) to pharmacological, physiological, and antigen-specific stimuli. However, preconditioning did appear to reduce the ability of monocytes and neutrophils to respond to activation signals, as indicated by lower levels of CD11b expression in stimulated cultures, and a significant increase in the basal production of IL-22 was also detected in PBMC cultured for 6 days following preconditioning. These alterations may reduce neutrophil and monocyte tissue infiltration and limit the inflammatory response during the early window of RIPC-induced protection and enhance tissue and wound repair several days later. A multivariate analysis confirmed that there was a significant difference in the response between the control and RIPC treatments and the main contributing factors were identified as changes in neutrophil and T cell activation, serum levels of MIP-1α and β, and production of IL-10 and IL-22 from PBMC cultured for 6 days. Overall, our results suggest that RIPC has a subtle but direct effect on the systemic innate immune response during the early window of protection in healthy volunteers, whereas the effects on the adaptive immune system seem to be considerably delayed. The changes detected following RIPC are likely to contribute to protection against ischaemia-reperfusion injury but not solely account for the extent of the beneficial effects of RIPC detected in animals. Our findings reinforce the safety profile of this intervention and have defined a number of immune parameters that are altered by preconditioning for focusing future research.</p>

Effect of remote ischaemic preconditioning on mortality and morbidity after non-cardiac surgery: meta-analysis

Background Remote ischaemic preconditioning (RIPC) has been shown to have a protective role on vital organs exposed to reperfusion injury. The aim of this systematic review was to evaluate the effects of non-invasive RIPC on clinical and biochemical outcomes in patients undergoing non-cardiac surgery Methods A systematic literature search of PubMed, EMBASE, Scopus, and Cochrane databases was carried out in February 2020. RCTs investigating the effect of non-invasive RIPC in adults undergoing non-cardiac surgery were included. Meta-analyses and trial sequential analyses (TSAs) were performed on cardiovascular events, acute kidney injury, and short- and long-term mortality. Results Some 43 RCTs including 3660 patients were included. The surgical areas comprised orthopaedic, vascular, abdominal, pulmonary, neurological, and urological surgery. Meta-analysis showed RIPC to be associated with fewer cardiovascular events in non-cardiac surgery (13 trials, 1968 patients, 421 events; odds ratio (OR) 0.68, 95 per cent c.i. 0.47 to 0.96; P = 0.03). Meta-analyses of the effect of RIPC on acute kidney injury (12 trials, 1208 patients, 211 events; OR 1.14, 0.78 to 1.69; P = 0.50; I2 = 9 per cent), short-term mortality (7 trials, 1239 patients, 65 events; OR 0.65, 0.37 to 1.12; P = 0.12; I2 = 0 per cent), and long-term mortality (4 trials, 1167 patients, 9 events; OR 0.67, 0.18 to 2.55; P = 0.56; I2 = 0 per cent) showed no significant differences for RIPC compared with standard perioperative care in non-cardiac surgery. However, TSAs showed that the required information sizes have not yet been reached. Conclusion Application of RIPC to non-cardiac surgery might reduce cardiovascular events, but not acute kidney injury or all-cause mortality, but currently available data are inadequate to confirm or reject an assumed intervention effect.

Can exercise training enhance the repeated remote ischaemic preconditioning stimulus on peripheral and cerebrovascular function in high-risk individuals?

Background Repeated exposure to remote ischaemic preconditioning (rIPC; short bouts of non-lethal ischaemia) enhances peripheral vascular function within 1 week; whereas, longer periods of rIPC (~ 1 year) may improve cerebral perfusion. Increasing the ‘dose’ of rIPC may lead to superior effects. Given the similarities between exercise and rIPC, we examined whether adding exercise to the rIPC stimulus leads to greater adaptation in systemic vascular function. Methods Nineteen individuals with increased risk for cardiovascular disease (CVD) were randomly allocated to either 8 weeks of rIPC (n = 9) or 8 weeks of rIPC + exercise (rIPC + Ex) (n = 10). rIPC was applied three times per week in both conditions, and exercise consisted of 50 min (70% heart rate max) of cycling 3 times per week. Peripheral endothelial function was assessed using flow-mediated dilation (FMD) before and after ischaemia–reperfusion (IR). Cerebrovascular function was assessed by dynamic cerebral autoregulation (dCA) and cerebrovascular reactivity (CVR), and cardio-respiratory fitness (VO2peak) using a maximal aerobic capacity test. Results FMD% increased by 1.6% (95% CI, 0.4, 2.8) following rIPC + Ex and by 0.3% (− 1.1, 1.5) in the only rIPC but this did not reach statistical significance (P = 0.65). Neither intervention evoked a change in dCA or in CVR (P > 0.05). VO2peak increased by 2.8 ml/kg/min (1.7, 3.9) following the rIPC + Ex and by 0.1 ml/kg/min (− 1.0, 1.4) following the rIPC only intervention (P = 0.69). Conclusion Combining exercise with rIPC across an 8-week intervention does not lead to superior effects in cerebrovascular and peripheral vascular function compared to a repeated rIPC intervention in individuals at risk of CVD.