Cardiovascular–kidney–metabolic syndrome drives coronary microvascular dysfunction in postmenopausal minipigs

Background – Cardiovascular-kidney-metabolic (CKM) syndrome is increasingly prevalent, its drivers, obesity, diabetes and chronic kidney disease being associated with increased cardiovascular morbidity and mortality. Coronary microvascular dysfunction (CMD) has been recognized as a central mechanism linking CKM with the adverse outcomes, especially in posmenopausal females with metabolic dysregulation. Purpose - Here we studied coronary microvascular function in postmenopausal miniswine with CKM. Methods – CKM was induced by combining diabetes mellitus (streptozotocin), hypercholesterolemia (high fat, high sugar diet), chronic kidney disease (renal embolization) in 5 adult, ovariectomized female minipigs (1.5-2 years old, ~40kg), while 11 healthy age- and weight-matched female minipigs on normal pig chow served as controls (Normal). At 6-months, coronary flow reserve (CFR, adenosine i.c.) was measured under anesthesia. Subsequently, we isolated coronary small arteries to study endothelial function and gene expression of vasoactive and pro- and anti-oxidant pathways. Results - 6 months of sustained hyperglycemia (17.5±1.0 in CKM vs 8.2±0.9 mmol/l in Normal, P<0.05 by t-test), hypercholesterolemia (15.4±2.0 vs 1.7±0.1 mmol/l, P<0.05), renal dysfunction (NGAL: 205±72 vs 67±5 ng/ml, P<0.05) and low progesterone levels (13±6 in CKM vs 92±30 nmol/l in Normal, P=0.1) were accompanied by systemic inflammation (TNFα: 62±7 vs 47±1 pg/ml, P<0.05). This was associated with microvascular dysfunction in vivo, shown by a significantly lower CFR in CKM compared to the Normal animals (3.4±0.4 vs 4.9±0.3, P<0.05). Isolated coronary small arteries from CKM animals showed impaired endothelium-dependent vasodilation to bradykinin (Fig. 1A) due to a loss of nitric oxide bioavailability, partly compensated for by activation of the cyclo-oxygenase (COX) pathway (Fig. 1B-C), while antioxidant treatment with Tempol significantly improved vasodilation. Consistent with the increased vasodilator influence of prostanoids, transcriptomic analyses of isolated coronary arterioles from CKM animals revealed an increased expression of COX1 (Fig. 2). Moreover, small, intermediate and big K+channel expression was higher in CKM, further potentiating the vasodilator effect of prostanoids. In contrast to the reduced NO-mediated vasodilation, eNOS expression was higher in CKM, accompanied by a reduced expression of the antioxidant enzyme SOD2, and higher expression of the pro-oxidant NOX-2 and SMPD2, confirming that oxidative stress compromised the vasodilator influence of NO. In support of this, CD31, sensor of oxidative stress was upregulated in CKM. Conclusion - Postmenopausal minipigs with CKM displayed a sustained microvascular endothelial pro-oxidative and pro-inflammatory state, resulting in severe coronary microvascular dysfunction evidenced by a significantly reduced CFR and NO bioavailability, partially compensated by activation of COX pathway.Fig. 1Fig. 2