Non-invasive Evaluation of “No Reflow” Phenomenon: Potential Role of Molecular Imaging

Reperfusion Injury Post Myocardial Infarction

The primary treatment for acute myocardial infarction (MI) involves early revascularization, and millions of individuals each year presenting with acute MI are successfully treated with early and effective interventional reperfusion strategies. However, despite this initial strategy to reduce the ischemic injury post MI, these patients are at significant risk for additional acute reperfusion injury and subsequent adverse LV remodeling and heart failure. ¹ In this context, a period of ischemia that is followed by reperfusion initiates a cascade of biological events within the injured myocardium, leading to reperfusion injury. This reperfusion injury can be associated with the “no reflow” phenomenon, which refers to the failure to restore perfusion to the microvasculature supplying the parenchyma of an organ after restoration of patency of the artery. ^{2, 3} In the heart no reflow occurs upon reperfusion following 30 to 90 minutes of coronary occlusion, and progresses over about 2 hours after the restoration of epicardial flow. This phenomenon has been attributed to microvascular injury and microvascular plugging resulting in microvascular obstruction (MVO) following restoration of epicardial flow with a percutaneous coronary intervention (PCI) generally in the setting of acute MI. The presence of MVO is associated with late post MI remodeling, heart failure and higher mortality. ^4–7

Detection of No-Reflow

In patients following I/R, “no reflow” can be identified immediately post a PCI angiographically by slow coronary filling (low TIMI score, high frame count) and low myocardial blush grade,⁸ or by demonstration of retrograde systolic flow and rapid deceleration of diastolic flow velocity with an intracoronary Doppler wire. Nuclear approaches have also been applied,⁹ but may be less reliable in this setting due to lower resolution and partial volume effects related to regional wall motion abnormalities post-MI. No reflow can be most effectively detected noninvasively with myocardial contrast echocardiography (MCE),⁴ or by late gadolinium enhanced cardiac MR imaging.^{6, 7} The MR defined no reflow area that has been termed MVO is characterized by a hypo-enhanced area within the hyper-enhanced infarct region.^{6, 10} This MR signature has become the preferred index for determination of MVO. The MR defined area of no reflow has been found to be associated with intramyocardial hemorrhage (IMH) in addition to complete MVO.¹¹ IMH occurs after severe microvascular injury due to loss of endothelial cell integrity and increases in vascular permeability, and is accompanied by edema and vascular compression.¹¹ The presence of intramyocardial hemorrhage post I/R has significant clinical implication and relevance to the study by Ozawa et al. in the current issue, which relied solely on hypo-perfusion on MCE to demonstrate no reflow.¹²

Important Role of Inflammation and Platelet Activation Post-I/R

Several studies have demonstrated that neutrophils exacerbate myocardial I/R injury.^{13, 14} Neutrophils contribute to myocardial I/R via several mechanisms; including microvascular plugging, release of vasoconstrictors, production of reactive oxygen species, release of proteolytic enzymes including matrix metalloproteinases, and platelet-activating factors. Neutrophils can cross the inflamed endothelium via an interaction between endothelium bound von Willebrand factor (VWF) and platelets mediated via platelet GPIbα.¹⁵

ADAMTS-13 and von Willebrand Factor: Dynamic Duo

A recent review by South and Lane highlight the important interactive role of VWF and ADAMTS-13.¹⁶ VWF is an adhesive glycoprotein that is essential for hemostasis that acts as a carrier for factor VIII and the capturing platelets at sites of vascular injury. ADAMTS-13 is a metalloproteinase that regulates the size and function of VWF. Severe deficiencies in ADAMTS-13 either congenital or acquired result in excesses of VWF and result in thrombocytopenic purpura (TTP). Moderate decreases in ADAMTS-13 activity can predispose to cardiovascular disease and increase risk for myocardial infarction in humans.¹⁷ Prior pre-clinical studies have demonstrated that ADAMTS-13 deficiency can lead to increased injury in murine models of ischemia/reperfusion using the C57BL6/J strain of mice.^{18, 19} In the study by Gandhi et al. that employed a murine model of 30 minutes of ischemia and 24 hours of reperfusion, ADAMTS-13 deficiency was associated with larger infarcts and increased neutrophil infiltration and myocyte apoptosis compared to wild-type controls.¹⁹ In contrast, VWF knockout mice demonstrated small infarcts and reduced neutrophil infiltration using the same model. These investigators also observed that myocardial I/R injury in the double knockout mice was similar to VWF −/− mice, suggesting that the exacerbation of I/R injury in the setting of ADAMTS-13 deficiency is VWF-dependent. De Meyer et al previously demonstrated using a similar closed-chest murine model of I/R that ADAMTS-13 deficient mice develop larger infarcts, and that administration of recombinant human ADAMTS-13 to wild-type mice was cardioprotective, and that the benefit was associated with a reduction in the infiltration of neutrophils and a reduction in platelet recruitment. ¹⁸ Similar findings were observed by Lindner and his colleagues using the same mouse strain and similar I/R protocol. The study by Ozawa et al employed both MCE perfusion imaging and molecular imaging to evaluate the role of microvascular endothelial-associated VWF and platelet adhesion in the no reflow phenomenon.¹² They observed increased mortality in ADAMTS-13 deficient mice, and reduced mortality in wild-type mice after delivery of recombinant ADAMTS-13. Molecular imaging demonstrated increased signal for platelets and VWF in the post ischemic risk area, and this was abolished by delivery of recombinant ADAMTS-13 early post reperfusion.

Findings in these C57BL6/J mice might not be directly translatable to humans, since these mice express a truncated and less active variant of ADAMTS-13. In addition, activation of VWF is shear mediated and arterial and venous flow rates are very different in mice and humans, and these differences could influence VWF function. ^{16, 19} There is also the theoretical concern for clinical translation that administration of recombinant ADAMTS-13 could induce a bleeding diathesis.

More importantly these observations in mice have not been confirmed by studies in a more clinically relevant porcine model and a pilot clinical study. Eerenberg et al. administered recombinant ADAMTS-13 to pigs immediately following I/R of the left circumflex coronary artery and demonstrated no reduction in infarct size or intramyocardial hemorrhage (IMH) as assessed with cardiac magnetic resonance (CMR) imaging, or formation of microthrombi or microvessels histologically as determined by CD31 staining compared to pigs given a vehicle control injection following reperfusion.²⁰ These pigs received unfractionated heparin, acetylsalicylic acid (ASA), and clopidogrel. Therefore, in the presence of dual anti-platelet therapy and heparin, which represents the standard of care, the administration of recombinant ADAMTS-13 did not reduce infarct size following I/R.

These same investigators also evaluated 49 patients post percutaneous intervention (PCI) that had CMR imaging 4 to 6 days post PCI, and demonstrated an early increase in VWF activity and antigen, and reductions of blood ADAMTS13 activity 4 and 7 days post PCI in only the subset of patients with CMR evidence of IMH.²⁰ However, VWF or ADAMTS-13 activity was not related to infarct size. These data significantly challenge the role of the balance of VWF and ADAMTS-13 as the cause of the “no reflow” phenomenon. It should be noted that these patients received anti-platelet therapy, including administration of ASA, prasugrel, and bivalrudin. Regardless, VWF and ADAMTS-13 may represent a surrogate marker of IMH and predict an increased risk of late post-MI complications.

Molecular Targeted Imaging

The important contribution of the study by Lindner and colleagues is the confirmation of a previously identified mechanism of the no reflow phenomenon with noninvasive molecular imaging. These investigators applied biotinylated lipid-shelled decafluorobutane microbubbles that were conjugated with ligands to target both VWF and platelets. In wild type mice treated early post reperfusion with recombinant ADAMTS-13 there was no increase in either the VWF or platelet signal within the post reperfusion ischemic risk area. The risk area signal was also less than that observed in both the untreated wild-type mice and the ADAMTS-13 knockout mice. Some regional differences were observed in the signal from the injured region compared to the salvaged post-ischemic region. These regional differences were not corrected for potential regional differences in flow and potential differences in microbubble delivery. However, their molecular imaging findings were confirmed by fluorescent immunohistochemistry for the presence of platelet adhesion within the risk area.

Conclusion

While the current study by Lindner and colleagues does not provide any new mechanistic insight into reperfusion injury and the no reflow phenomenon, their study does provide an important illustration of how targeted molecular imaging can noninvasively assess mechanisms of injury, and theoretically be used to guide and evaluate therapeutic interventions. Unfortunately, the observed benefit of delivery of recombinant ADAMTS-13 post-reperfusion in these murine models has not been confirmed by large animal studies or an early clinical observational trial. It would be important to apply this novel ultrasound-based targeted molecular and physiological imaging approach in these other more clinically relevant settings. The ultrasound imaging technology is uniquely suited for addressing this problem, and could be potentially performed at the bedside. Reperfusion injury post PCI in the setting of acute MI remains a major clinical problem and application of this novel imaging technology could help solve this challenging problem.