The multifunctional Ca²⁺/calmodulin-dependent kinase IIδ (CaMKIIδ) regulates arteriogenesis in a mouse model of flow-mediated remodeling

Abstract

Objective: Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca²⁺/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.

Methods and results: Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ-/-) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ-/- mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ-/- mice normalized flow-mediated remodeling.

Conclusion: CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.

Figure 1. CaMKIIδ is required for flow-mediated remodeling.

(A) Diagram of experimental approach. Arteriogenesis is induced in the right common carotid artery (CCA) after left CCA ligation. (B) Representative H&E-stained right carotid arteries of WT and CaMKIIδ−/− mice at baseline (day 0) and day 28 post-left carotid ligation. Scale bar = 200 µm. (C) Quantification of the perimeter of the internal (IEL) and external elastic lamina (EEL) (n = 6 for day 0 and n = 10 for days 14 and 28). (D) Ultrasound cross-sectional images of the right common carotid artery 28 days after left carotid ligation. The insets demonstrate color Doppler flow in the right carotid. No flow was detected in the left common carotid. (E) Quantification of the anterior-posterior diameter of right common carotid arteries of WT and CaMKIIδ−/− mice (n = 10 per genotype, experiments are independent of (B) and (C)). *p<0.05 compared to baseline; **p<0.05 compared to WT.

Figure 2. CaMKII is upregulated and activated in arteriogenesis.

(A) CaMKII activity in right carotid arteries from WT and CaMKIIδ−/− mice 14 days after left carotid ligation. (B) Immunolabeling for total CaMKII (green; SM–actin red; nuclei blue) in WT and CaMKIIδ−/− right carotid artery sections on day 14 after ligation. Arrow, adventitia; arrowhead, endothelium. (C) Fold change in mRNA expression of CaMKIIδ and γ in right carotids isolated from WT and CaMKIIδ−/− mice by quantitative RT-PCR. (D) Immunolabeling for oxidized (ox-CaMKII, green, left panel) and phosphorylated CaMKII (p-CaMKII, green, right panel) in WT right carotid artery sections (SM–actin red; nuclei blue). Arrowheads indicate single cells with p-CaMKII labeling. Scale bar = 30 µm.

Figure 3. ROS and oxidative activation of CaMKII in the right carotid artery after left carotid ligation.

(A) NADPH oxidase subunit p47 expression in WT and CaMKIIδ−/− right carotid arteries at baseline and on day 14 after left carotid ligation. (B) ROS production in the vascular wall of WT and CaMKIIδ−/− right carotid arteries at baseline and on day 14 after left carotid ligation. (C) Immunolabeling for ox-CaMKII (green; SM-actin, red; nuclei, blue) in WT and CaMKII MV carotid artery sections at baseline and 14 days post-ligation. (D) Representative H&E-stained right carotid arteries of WT and CaMKII MV mice. (E) Quantification of the perimeter of the IEL and EEL (n = 6 for day 0 and n = 10 for day 14 and 28). *p<0.05 compared to baseline; **p<0.05 compared to WT. Scale bar = 30 µm in C, 100 µm in D.

Figure 4. Adventitial macrophages express CaMKII.

(A) Double labeling of adventitial macrophages from WT mice for total CaMKII (green) and the macrophage marker Mac-3 (red) on day 7 after ligation. Nuclei were stained with DAPI (blue). Arrowheads indicate macrophages. Scale bar = 30 µm. (B) Quantitative RT-PCR for CaMKIIδ and γ in WT and CaMKIIδ−/− BMMs before and after treatment with 1 µg/ml LPS for 6 hr. (C) Left panel, quantification of the number of Mac-3-labeled macrophages in sections of right WT and CaMKIIδ−/− carotid arteries after left carotid ligation. Right panel, quantitative RT-PCR for the macrophage marker F4/80 at day 7 post-ligation relative to baseline (p = 0.152 between genotypes). (D) Quantitative RT-PCR for IL-6, IL-1β and TNF-α in WT and CaMKIIδ−/− BMMs at baseline and 6hr after LPS treatment (1 µg/ml). (n = 9 mice per group) *p<0.05 compared to baseline, **p<0.05 compared to WT.

Figure 5. CaMKIIδ promotes MMP9 expression in flow-mediated remodeling.

(A) MMP9 immunolabeling (green; SM-actin, red; To-ProIII, blue) in right carotid artery sections from WT and CaMKIIδ−/− mice before and 7 days after left carotid ligation. Scale bar = 100 µm. (B) Quantification of MMP9 staining intensity. (C) Magnification of MMP9 adventitial labeling (left panel, green; SM-actin, red) and Masson Trichrome staining (right panel) in WT right carotid artery section 7 days post-ligation. Scale bar = 50 µm. (D) Adventitial MMP9 (red) and macrophage marker F4/80 (green) double-labeling in right carotids from WT mice. Nuclei were stained with To-ProIII (blue); * indicates co-localization. (E) Quantitative RT-PCR for MMP9 in BMMs 6 hr after addition of LPS (1 µg/ml, p = 0.053). (F) Quantitative RT-PCR for MMP9 in right carotid arteries 1 and 7 days after left ligation. (G) MMP9 activity in right carotid artery homogenates at baseline and 7 days after ligation. *p<0.05 compared to day 0; **p<0.05 compared to WT.

Figure 6. Transplantation of WT bone marrow cells into CaMKIIδ−/− mice restores arteriogenesis.

(A) Quantification of the anterior-posterior diameter of right carotid arteries of WT or CaMKIIδ−/− mice transplanted with WT BM (n = 6 for WT and n = 10 for CaMKIIδ−/− mice, *p<0.05 compared to day 0). (B) Representative H&E-stained WT and CaMKIIδ−/− right carotid arteries after transplantation of WT bone marrow. Carotid artery ligations were performed 8 weeks after BM transplantation. Scale bar = 200 µm (C) Quantification of the perimeter of the IEL and EEL (n = 6 for WT and n = 10 for CaMKIIδ−/− mice).

Figure 7. CaMKIIδ deletion does not decrease VSMC proliferation or endothelial expression of VCAM-1 or CD54 (ICAM-1).

(A) Representative image of BrdU-labeling of WT right common carotid artery 28 days post-ligation. Inset: neointima in the left carotid artery of the same mouse as positive control. Scale bar = 100 µm. (B) Quantification of medial cells from right carotid artery sections labeled for SM-actin and nuclei (To-ProIII). (C) Left panels, representative immunofluorescent images of VCAM-1 (green) in the right common carotid artery 7 days post-ligation (SM-actin, red; nuclei, blue). Scale bar = 100 µm. Right panel, quantification of endothelial VCAM-1 labeling. *p<0.05 compared to baseline; **p<0.05 compared to WT. (D) left panels, representative immunofluorescent images of CD45 (ICAM-1) (green) in the right common carotid artery 7 days post-ligation (SM-actin, red; nuclei, blue). Right panel, quantification of endothelial CD45 labeling (n = 5).