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NADPH oxidase inhibition opposes pro-oxidant and apoptotic signaling and partially rescues impairment in coronary microvascular vasorelaxation in diabetic models
*Debolina Banerjee MD, *Ju-woo Nho , *Hang Xing , *Janelle Li , *Hanisah Iddrisu , *Meghamsh Kanuparthy MD, Frank Sellke MD, *Jun Feng
Surgery, Brown University/Rhode Island Hospital, Providence, RI

Objective: Cardioplegic ischemia/reperfusion (I/R) injury continues to pose substantial challenges during postoperative recovery following cardiac surgery, with diabetic patients particularly susceptible to adverse cardiac events. Overactivation of NAPDH oxidase (NOX) has been implicated in vascular endothelial barrier dysfunction following cardioplegic I/R through stimulating downstream production of reactive oxygen species and ultimately cellular apoptosis. Mechanistic studies have stipulated the predominant role of NOX in diabetic coronary microvascular complications We aimed to investigate NOX inhibition as a viable therapeutic in modulating apoptosis and oxidative stress in diabetic and nondiabetic human endothelial cells as well as murine coronary microvessels subjected to simulated cardioplegic I/R.
Design: Human coronary arterial endothelial cells (HCAECs, n = 6 per group) were cultured in normoglycemic (C) and hyperglycemic (D) conditions, subjected to 3 hours of hypoxia followed by 2 hours of reoxygenation alone (DH and CH groups), or subjected to aforementioned hypoxia/reoxygenation followed by treatment with NOX inhibitor apocynin (DHT and CHT groups). Whole-cell protein purification and immunoblotting were used to assess molecular signaling. Cardioplegia hypoxia/reoxygenation model used for murine studies, treatment group received apocynin (30mg/kg/d ip) for 4 weeks.
Setting: Basic science laboratory.
Patients or participants: HCAECs, 18 12-16-week-old male mice (6 C57BL/6J nondiabetic, 12 Cg-Dock7m +/+ Leprdb/J diabetic.
Interventions: Hyperglycemia, hypoxia/reoxygenation, apocynin.
Main Outcome Measures: Ex vivo coronary arteriolar response to ADP, NS309, SNP; immunoblotting results.
Results: Coronary microvessels from diabetic mice subjected to simulated cardioplegic I/R had significantly impaired vasorelaxation to both endothelium-dependent dilator ADP and SK channel activator NS309 when compared to nondiabetic counterparts. Apocynin treatment rescued this effect in diabetic mice, with 50% recovery in vasorelaxation in response to ADP and NS309, but not endothelium-independent dilator SNP. In HCAECs, apocynin treatment was associated with decreased markers of oxidative stress. Phosphorylated endothelial nitric oxide synthase (p-eNOS) increased with I/R (p = 0.005) but reversed with apocynin (p = 0.004) in C/CH/CHT. Apocynin significantly decreased p-eNOS in DHT versus D or DH (p <0.0001). These trends were reflected in p-eNOS:eNOS ratios for C/CH/CHT (p <0.03) and D/DH/DHT (p <0.0001) groups. Similarly, apocynin antagonized NOX 1 signaling in C/CH/CHT and D/DH/DHT. NOX inhibition decreased pro-apoptotic phosphorylated FOXO (p-FOXO) in D/DH/DHT with little effect in C/CH/CHT groups.
Conclusions: NOX inhibition partially rescues impairment in coronary microvascular vasorelaxation and opposes pro-oxidant and apoptotic signaling in diabetic models. Cardioprotective strategies that incorporate NOX inhibition warrant further investigation.


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