Coronary Microvascular Smooth Muscle‐Endothelial Cell Communication and Notch 3 Signaling in Type 2 Diabetes

Abstract

The Notch signaling pathway, via cell‐cell communication, is critical during normal vascular development and homeostasis, and it is known to regulate vascular smooth muscle (VSMC) phenotype, including proliferation. The current study tested the hypothesis that VSMC‐endothelial cell (EC) Notch 3/Jagged 1 signaling is aberrant to account for adverse remodeling and stiffness observed in type 2 diabetic coronary resistance microvessels (CRMs). Low‐passage primary coronary VMSCs were isolated from normal and type 2 diabetic db/db mice and were either mono‐ or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively (n=4–6 per group). In separate experiments, normal (n=3) and diabetic db/db CRMs (n=3) were isolated, mounted, and perfusion pressure fixed on a pressure myograph. The size and frequency of fenestrae in the internal elastic laminas of intact CRMs were assessed using Alexa Fluor 633 hydrazide on a confocal microscope. Coronary VSMCs isolated from db/db mice expressed higher Notch 3 mRNA (2.12±0.10 vs. 1.00±0.20; p<0.01) compared to normal. Co‐culture of coronary VSCMs with hCMECs caused a robust induction of Notch 3 expression in normal VSMCs (3.67±0.60 vs. 1.00, p<0.001), which was exacerbated in coronary VSMCs isolated from db/db mice (5.52±0.51 vs. 3.67±0.60, p<0.05). Alpha‐smooth muscle actin (α‐SMA) trended lower in db/db coronary VSMCs (p=0.07), while co‐culture with hCMECs did not alter α‐SMA expression. Diabetic hCMECs expressed lower Jagged 1 mRNA compared to normal (0.58±0.04 vs. 1.00±0.07; p<0.01); co‐culture of hCMECs and coronary VSMCs caused a robust reduction in Jagged 1 expression in normal cells (0.63±0.06 vs. 1.00±0.07, p<0.001), while no further reduction was observed in co‐cultured diabetic cells. The number of fenestrae in the internal elastic lamina was reduced in diabetic CRMs (0.028±0.005 vs. 0.040±0.001 fen/μm2, p<0.05), while the size of the fenestrae remained unchanged. These data show that Notch3/Jagged 1 expression and VSMC differentiation are altered in diabetic coronary resistance microvessel cells, and VSMC‐EC contact via co‐culture induces differential dysregulation of Notch3/Jagged1 signaling. The data further show a reduction in the number of fenestrae in db/db CRMs, suggesting that the opportunity for Notch3/Jagged1 signaling to occur in vivo may be reduced to contribute to adverse remodeling and stiffness observed in those blood vessels.Support or Funding InformationAHA 15UFEL22760002 to MK; NIH K99HL116769 and NCH to AJT.