The Role of Nitric Oxide during Sonoreperfusion of Microvascular Obstruction

Abstract

Rationale: Microembolization during PCI for acute myocardial infarction can cause microvascular obstruction (MVO). MVO severely limits the success of reperfusion therapies, is associated with additional myonecrosis, and is linked to worse prognosis, including death. We have shown, both in in vitro and in vivo models, that ultrasound (US) and microbubble (MB) therapy (termed “sonoreperfusion” or “SRP”) is a theranostic approach that relieves MVO and restores perfusion, but the underlying mechanisms remain to be established.Objective: In this study, we investigated the role of nitric oxide (NO) during SRP.Methods and results: We first demonstrated in plated cells that US-stimulated MB oscillations induced a 6-fold increase in endothelial nitric oxide synthase (eNOS) phosphorylation in vitro. We then monitored the kinetics of intramuscular NO and perfusion flow rate responses following 2-min of SRP therapy in the rat hindlimb muscle, with and without blockade of eNOS with LNAME. Following SRP, we found that starting at 6 minutes, intramuscular NO increased significantly over 30 min and was higher than baseline after 13 min. Concomitant contrast enhanced burst reperfusion imaging confirmed that there was a marked increase in perfusion flow rate at 6 and 10 min post SRP compared to baseline (>2.5 fold). The increases in intramuscular NO and perfusion rate were blunted with LNAME. Finally, we tested the hypothesis that NO plays a role in SRP by assessing reperfusion efficacy in a previously described rat hindlimb model of MVO during blockade of eNOS. After US treatment 1, microvascular blood volume was restored to baseline in the MB+US group, but remained low in the LNAME group. Perfusion rates increased in the MB+US group after US treatment 2 but not in the MB+US+LNAME group.Conclusions: These data strongly support that MB oscillations can activate the eNOS pathway leading to increased blood perfusion and that NO plays a significant role in SRP efficacy.

Ultrasound-Responsive Cavitation Nuclei for Therapy and Drug Delivery

“…The majority of these bio-effects have been observed in in vitro models using largely non-endothelial cells and may therefore not be directly relevant to in vivo tissue, where intravascular micron-sized cavitation nuclei will only have contact with endothelial cells and circulating blood cells. On the other hand, the mechanistic studies by Belcik et al (2015Belcik et al ( , 2017 and Yu et al (2017) do reveal translation from in vitro to in vivo. In these studies, ultrasound-activated microbubbles were found to induce a shear-dependent increase in intravascular adenosine triphosphate (ATP) from both endothelial cells and erythrocytes, an increase in intramuscular nitric oxide and downstream signaling through both nitric oxide and prostaglandins, which resulted in augmentation of muscle blood flow.…”

“…In these studies, ultrasound-activated microbubbles were found to induce a shear-dependent increase in intravascular adenosine triphosphate (ATP) from both endothelial cells and erythrocytes, an increase in intramuscular nitric oxide and downstream signaling through both nitric oxide and prostaglandins, which resulted in augmentation of muscle blood flow. Ultrasound settings were similar, namely, 1.3 MHz, mechanical index (MI) 1.3 for Belcik et al (2015Belcik et al ( , 2017 and 1 MHz, MI 1.5 for Yu et al (2017), with MI defined as MI = P À / ffiffi ffi f p , where P_ is the derated peak negative pressure of the ultrasound wave (in MPa) and f the center frequency of the ultrasound wave (in MHz).…”

Role of Oxidative Stress in Reperfusion following Myocardial Ischemia and Its Treatments

“…In addition, due to the antiplatelet aggregation and vasodilation effects of NO [ 184 ], it may reduce microvascular obstruction following reperfusion. Similar therapeutic effects may be achieved through microbubble oscillations, which can increase blood perfusion through activating the eNOS pathway and releasing NO [ 185 ].…”

The effect of ultrasound pulse length on microbubble cavitation induced antibody accumulation and distribution in a mouse model of breast cancer

“…Interestingly, short and long pulses have been used in the BBB literature and we therefore designed our experimental conditions to explore different pulse lengths, but with the same total acoustical power and number of cycles. We were expecting differences since it is known that MB cavitation activity is higher with long pulses compared to shorter pulses 35 - 37 . Within a very large parameter space for US pulse design, spanning from moderate (100μs) 16 to very long (2-10ms) pulse length in recent literature 15 , 17 , we opted for three pulses composed of the same total number of cycles but separated in trains of 1x 5000 cycles, 125 x 40 cycles and 500 x 10 cycles, with the objective of determining if pulse length played an role in UTMC antibody therapy.…”