This study gives first evidence that APOSEC effectively reduces MVO in a clinically relevant ischemia/reperfusion AMI model. This finding was associated with an improvement in the myocardial blush grade and corrected TIMI frame count, two clinically established parameters of microvascular patency. Moreover, resolution of ECG alterations during experimental occlusion and reperfusion were mediated by treating animals with APOSEC. The impact of APOSEC on two major contributors of MVO was tested in vitro. Co-incubation of platelets and APOSEC led to an increase of phosphorylated VASP, consecutively inhibiting platelet aggregation in vitro. Treating HUVEC with APOSEC resulted in an induction of iNOS and p-eNOS. Additionally, direct vasodilatory effects of APOSEC were shown in myographical evaluations of isolated coronary arterial rings.
For a long time, beneficial effects in stem cell therapy were contributed solely to cellular mediated mechanism. Recently, this concept was challenged by works showing that paracrine signalling may be a significant additional mode of action [19
]. The importance of releasing pro-angiogenic and cytoprotective factors during AMI has already been shown for mesenchymal as well as for bone marrow derived stem cells [3
]. We have recently expanded the concept of regeneratory, paracrine factors derived from stem cell, by showing that the secretome of apoptotic PBMC attenuates myocardial infarction [39
]. The major advantage of PBMC over stem cells is that they are a lot easier to access. Although secretome of stem cells and PBMC both mediate similar effects, their secreted factors slightly differ. In a protein chip array study Wollert and colleagues showed that out of 174 secreted factors, 25 factors were present in higher concentrations in bone marrow supernatants, and ten factors were found in higher concentrations in peripheral blood leucocytes [36
]. To the best of our knowledge, our group was first to utilize the potential of paracrine factors derived from PBMC in an experimental AMI setting. Consequently, we have addressed features of APOSEC relevant for microvascular obstruction in this subsequent study.
After re-establishing blood flow in the occluded epicardial vessel, the integrity of the microcirculation in the vicinity of the post-ischemic myocardium is pivotal for a patient’s prognosis. An open microvasculature was shown to supply infarct related myocardium with blood and avoiding myocyte necrosis [10
]. It is of utmost importance to maintain this residual blood flow within the AAR, since there is sufficient in vitro and in vivo evidence of viable myocardium hours to days after coronary occlusion [32
]. If the preservation of microvascular flow fails, viable myocardium is gradually lost. It is a currently accepted notion that platelets are causative for microvascular dysfunction by releasing vasoconstrictive substances [21
], by forming microemboli [28
] or by intravasal thrombus formation in the microcirculation [7
]. Moreover, experimental evidence indicates that the detrimental effect of platelets is dependent on their activation status [64
]. Relevant to MVO are the observations of Barrabes et al. [8
], who showed in a porcine AMI model that ischemic injury triggers macro- and microvascular platelet deposition even in distant areas not related to the occluded coronary artery. This leads to impairment in coronary flow reserve and contractile function. With advances in understanding the pathophysiology of microvascular malperfusion, different therapeutic strategies inhibiting platelet function have been proposed. However, to date only the application of monoclonal antibodies blocking GPIIb–IIIa receptor improved microvascular flow and subsequently reduced infarct size in animal models [37
]. This effect could be confirmed in double-blind randomized trials which have led to a class IIA recommendation of use of anti GP IIb/IIIa in the ACC/AHA guidelines [5
Currently there is no standard in measuring microvasculature dysfunction in vivo. Several techniques including coronary angiography, contrast echocardiography, and MRI are used clinically and experimentally to describe MVO. Each of these techniques measures slightly different biological and functional parameters [9
]. We therefore decided to confirm our MRI data with cardiac catheterization measurements. A low TIMI frame count indicates a sufficient blood flow in the small vessels; on the other hand a high TIMI frame count is associated with microvascular occlusion [18
]. The angiographic myocardial blush grade is a standard method to clinically assess myocardial tissue perfusion [24
]. It has a direct impact on patients’ prognosis since a persistently abnormal myocardial blush grade was shown to result in reduced functional parameters in the long-term [30
No-reflow phenomenon is known to be associated with persistent ST-elevation and ventricular arrhythmias [12
]. About 25 % of patients ST-segment abnormalities persist even though coronary blood flow has been restored. Therefore, we sought to determine whether APOSEC has an effect on ECG alterations during AMI. As shown in Table , infusion of APOSEC led to a normalization of ST segment alterations in the majority of treated animals. In addition, arrhythmic episodes were lower in the APOSEC group during occlusion and reperfusion.
The beneficial effects of APOSEC on MVO in our porcine in vivo AMI model are in line with in vitro data obtained after exposure of porcine platelets to APOSEC. Co-incubation of platelets with APOSEC prevented platelet aggregation triggered by collagen. Based on these findings further experiments were performed with human platelets and similar effects could be observed. The addition of TRAP-6 at a final concentration of 10 μM and ADP at a concentration of 50 μM caused platelets to fully aggregate and this aggregation was effectively impaired by preincubation of platelets with APOSEC. Interestingly, APOSEC derived from PBMCs isolated from diabetic and heart insufficiency patients triggered the same effects compared to APOSEC obtained from healthy patients.
Platelet surface P-selectin is considered to be the “gold standard” marker of platelet activation and was significantly reduced after preincubation of purified platelets with APOSEC [43
]. This finding was further supported by reduced platelet surface markers CD63 and CD40L and lower concentrations of sCD40L, sP-selectin, and TSP-1 in the supernatant of APOSEC exposed platelets.
A recent paper by Köhler et al. [35
] has provided profound evidence that the phosphorylation state of VASP is crucially important for the extent of myocardial ischemia/reperfusion injury. Increased intra-platelet phosphorylated VASP was shown to prevent platelet activation and platelet-neutrophil complex formation during AMI. These findings were meticulously confirmed with VASP knock-out animals, bone marrow chimeric animals and a platelet transfer model. Therapeutic augmentation of phosphorylated VASP using a guanylyl cyclase activator was shown to be effective in a rodent animal model [54
]. We consequently addressed the question whether APOSEC is capable to induce VASP phosphorylation in platelets. Indeed, we were able to show that APOSEC led to an increase of phosphorylated VASP, and these effects of APOSEC could be observed in the absence as well as in the presence of submaximal effective concentrations of prostaglandin E1
Besides platelet activation and aggregation, endothelial dysfunction in the small coronary vasculature is another major component in the pathophysiology of the no-reflow phenomenon. During reperfusion the endothelium is injured by oxygen free radicals resulting in an impaired endothelium-dependent vasodilation [55
]. Besides, aspirates from coronary arteries obtained during PCI were shown to contain vasoconstrictor factors [34
]. The concept of increased vasomotor tone in the area of MVO is supported by several clinical trials, testing different vasodilators during PCI. Currently, adenosine, verapamil or nitroprusside are a recommended therapeutic option for the treatment of no-reflow [25
]. Since “classical” vasodilatory drugs have been proven beneficial in the setting of no-reflow, we investigated whether APOSEC has also an effect on the vasomotor tone. In plasma samples obtained after AMI induction, systemic levels of vasodilatory mediators were heightened. In line with this finding we were able to show that HUVEC upregulated iNOS and p-eNOS expression after application of APOSEC. Besides these long-term effects on NO synthases, also a direct vasodilatory impact of APOSEC on isolated coronary vessel rings was observed, which was independent of NOS activity (Fig. , Suppl. Fig. 2).
Despite the effects of APOSEC on expression and activation of nitric oxide synthases, some immediately occurring effects might (also) be caused directly by biologically active compounds residing in APOSEC. In this regard, the identification of significant amounts of nitrite/nitrate in APOSEC preparations might be of central importance. As APOSEC is extensively dialyzed we can exclude the possibility that NO decomposition products nitrite and nitrate are present in APOSEC. Therefore, we consider it safe to conclude that protein adducts of nitric oxide represent a biologically active ingredient of APOSEC. The NO-axis has been shown to mediate cardioprotective signalling [27
] and locally liberated NO might be responsible for some of the immediate effects of APOSEC, especially those we could observe in experiments dealing with vascular tension and platelet activation. Specifically, such a mechanism would be in line with APOSEC-mediated vasodilation that occurs in L-NAME treated coronary rings and the finding that APOSEC enhances VASP phosphorylation even when applied alone (i.e., in the absence of prostaglandin E1
For the current study APOSEC was produced and tested in allogeneic fashion, hence all experiments were performed with APOSEC obtained from (genetically non-identical) donors of the same species. In order to extend to the clinical reality we also obtained APOSEC from diabetic and heart failure patients. As shown in Suppl. Figs. and b concentrations of reference cytokines and results of functional assays (platelet aggregation, iNOS, p-eNOS induction in HUVEC) were comparable in APOSEC derived from healthy and diseased patients. Consequently, these data suggest autologous (“autotransplantation” of APOSEC derived from a diseased patient) as well as allogeneic source (APOSEC derived from healthy donors, similar to plasma derivatives) might be feasible options for patients suffering of hypoxia induced ischemic conditions. In respect to planned autologous and allogeneic APOSEC production strict regulatory prerequisites (e.g., virus inactivation, potency assays, and mandated GMP facilities) have to be met in order to reach human clinical trials.