The adenylate cyclase type 9 (ADCY9) gene appears to determine atherosclerotic outcomes in patients treated with dalcetrapib. In mice, we recently demonstrated that Adcy9 inactivation potentiates ...endothelial function and inhibits atherogenesis. The objective of this study was to characterize the contribution of ADCY9 to the regulation of endothelial signalling pathways involved in atherosclerosis.
We show that ADCY9 is expressed in the endothelium of mouse aorta and femoral arteries. We demonstrate that ADCY9 inactivation in cultured endothelial cells paradoxically increases cAMP accumulation in response to the adenylate cyclase activators forskolin and vasoactive intestinal peptide (VIP). Reciprocally, ADCY9 overexpression decreases cAMP production. Using mouse femoral artery arteriography, we show that Adcy9 inactivation potentiates VIP-induced endothelial-dependent vasodilation. Moreover, Adcy9 inactivation reduces mouse atheroma endothelial permeability in different vascular beds. ADCY9 overexpression reduces forskolin-induced phosphorylation of Ser157-vasodilator-stimulated phosphoprotein (VASP) and worsens thrombin-induced fall of RAP1 activity, both leading to increased endothelial permeability. ADCY9 inactivation in thrombin-stimulated human coronary artery endothelial cells results in cAMP accumulation, increases p-Ser157-VASP, and inhibits endothelial permeability. MLC2 phosphorylation and actin stress fibre increases in response to thrombin were reduced by ADCY9 inactivation, suggesting actin cytoskeleton regulation. Finally, using the Miles assay, we demonstrate that Adcy9 regulates thrombin-induced endothelial permeability in vivo in normal and atherosclerotic animals.
Adcy9 is expressed in endothelial cells and regulates local cAMP and endothelial functions including permeability relevant to atherogenesis.
BACKGROUND:Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and ...atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined.
METHODS:Adcy9-inactivated (Adcy9) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtgAdcy9 and CETPtgAdcy9), were submitted to an atherogenic protocol (injection of an AAV8 adeno-associated virus serotype 8 expressing a PCSK9 proprotein convertase subtilisin/kexin type 9 gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated.
RESULTS:Adcy9 mice had a 65% reduction in aortic atherosclerosis compared to WT (P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9 mice compared to WT animals (P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9 mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9 (P<0.01 for all). Aortic endothelium from Adcy9 mice allowed significantly less adhesion of splenocytes compared to WT (P<0.05). Adcy9 mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume (P<0.01 for both). Feed efficiency was increased in Adcy9 compared to WT mice (P<0.01), which was accompanied by prolonged cardiac RR interval (P<0.05) and improved nocturnal heart rate variability (P=0.0572). Adcy9 inactivation–induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtgAdcy9 mice (P>0.05 versus CETPtgAdcy9).
CONCLUSIONS:Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.
Abstract only Adenylate cyclase (AC) activity in vessels is associated with vasodilation. The AC family includes 10 members of which AC9 is the least studied. We recently demonstrated that Adcy9 ...inactivation in mice protects from atherosclerosis in the aorta and potentiates endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) in femoral arteries (FA). Our objectives were 1) to determine Adcy9 expression in FA; 2) investigate endothelial signalling pathways associated with Adcy9 inactivation-induced potentiation of ACh-induced EDV; and 3) to assess the effect of Adcy9 inactivation on EDV during flow-mediated dilation (FMD). FA were isolated from 8 to 13 week-old Adcy9- inactivated ( Adcy9 Gt ) and wild-type (WT) littermate mice. Adcy9 expression was studied in WT FA tissue section by in situ hybridization (ISH) using Adcy9 and negative control probes. Diameter of pressurized (80 mmHg) FA (internal diameter 288±3 μm) was studied in response to ACh (n=7-14) and shear stress (n=12) up to 20 dynes/cm 2 after preconstriction with phenylephrine. To study the role of endothelial signalling pathways in response to ACh-induced EDV, FA were incubated in absence or presence of inhibitors for nitric oxide (NO) synthase (L-NNA 0.1 μM), cyclooxygenase (meclofenamate 1 μM) and endothelial hyperpolarization (TRAM-34 10 μM plus apamin 0.1 μM). ISH with Adcy9 probe resulted in numerous dots in the vascular wall of the FA. Meclofenamate and the combination of TRAM-34 plus apamin decreased maximal (E max ) EDV to ACh, respectively, by 40 and 17% in Adcy9 Gt (P<0.01) but had no effect in WT mice. L-NNA reduced E max to ACh by 26% in WT (P<0.01) and 50% in Adcy9 Gt (P<0.01). Adcy9 inactivation tended to increase FMD to shear stress ranging from 6 to 20 dynes/cm 2 : At 10 dynes/cm 2 , FMD was increased from 25.4±4.9% in WT to 37.4±4.9% in Adcy9 Gt mice (P=0.083, n=12 per group). In conclusion, our data show that FA express Adcy9 and that Adcy9 inactivation increases the effects of endothelial NO, hyperpolarization and cyclooxygenase pathways in EDV. A numerical increase in FMD in Adcy9 Gt compared to WT confirms the potentiating effect of Adcy9 inactivation on EDV. The potentiation of endothelial dilatory pathways appears to be associated with the anti-atherosclerotic effects of Adcy9 inactivation.
Abstract only Polymorphisms in the ADCY9 gene, coding for adenylate cyclase type 9, determine atherosclerotic cardiovascular responses to the CETP inhibitor dalcetrapib in patients. ADCY9 is broadly ...expressed and involved in various immune cell functions and inflammatory responses. Our objective is to determine the role of ADCY9 in the development of atherosclerosis in the absence of CETP. We used 8-12 week-old wild-type (WT, n=25) or Adcy9 -inactivated ( Adcy9 Gt , n=21) male mice. To induce hypercholesterolemia and atherosclerosis, mice were infected with an adeno-associated virus coding for a gain-of-function mutant of Pcsk9 ( Pcsk9 D377Y ) and were fed an atherogenic high-cholesterol diet for 16 weeks in absence or presence of dalcetrapib treatment (200 mg/kg/day). Percent atherosclerotic lesion area (PALA) in the whole aorta was quantified using en face Oil Red O plaque staining. We used VCAM-1 immunofluorescence staining (n=6 per group) in atherosclerotic lesions of the aortic root to quantify inflammation. We also measured aortic root accumulation of macrophages and vascular smooth muscle cells by immunofluorescence imaging of CD68 and smooth muscle actin (SMA), respectively, in plaque lesions. We also quantified blood leukocytes in normocholesterolemic WT and Adcy9 Gt mice by flow cytometry analysis of CD45 + cells. In hypercholesterolemic animals, Adcy9 Gt mice showed a 60% decrease in atherosclerotic lesions (PALA: 2.7±0.5%) compared to WT mice (6.5±0.7%, P<0.01). Dalcetrapib treatment, in these mice without CETP, did not modify significantly the reduction (63%) of PALA in Adcy9 Gt mice (2.9±0.4%) compared to WT (7.6±1.6%, P<0.01). Macrophage content was reduced by 55% from 17.8±2.8% in WT to 7.2%±1.9% in Adcy9 Gt mice (P<0.05), while SMA staining was similar. VCAM-1 expression also tended to be decreased, from 5.7±3.3% in WT to 2.0±0.4% in Adcy9 Gt mice (P=0.0598). CD45 + blood leukocytes were reduced by 35.4% in Adcy9 Gt compared to WT normocholesterolemic mice (P<0.01). Adcy9 inactivation in mice (without CETP) results in large reductions of atherosclerosis and plaque macrophage and in decreased VCAM-1 expression. These results support the ADCY9 genotype-dependent clinical effects of dalcetrapib.
Abstract only Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the cholesteryl ester transfer protein (CETP) inhibitor dalcetrapib on cardiovascular events, ...atherosclerosis imaging and body weight variation. The underlying mechanisms responsible for the interactions between ADCY9 and CETP have not yet been determined. Adcy9 -inactivated ( Adcy9 Gt/Gt ) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETP Gt and CETP WT ), were submitted to an atherogenic protocol (injection of an AAV8 expressing a PCSK9 gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, cell adhesion, vasorelaxation, telemetry and adipose tissue MRI were evaluated. Adcy9 Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P <0.01). CD68-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9 Gt/Gt mice compared to WT animals ( P <0.05 for both). Adcy9 inactivation did not change counts of blood monocytes and their subsets. Splenocytes showed reduced adhesion to native aortic endothelium from Adcy9 Gt/Gt mice ( P <0.05 vs WT). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9 Gt/Gt mice (versus WT, P <0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase and endothelial-dependent hyperpolarization pathways all contributed to the improvement of vasodilatation in Adcy9 Gt/Gt versus WT ( P <0.01 for all). Adcy9 Gt/Gt mice gained more weight than WT with the atherogenic diet, and this was associated with an increase in whole body adipose tissue volume ( P <0.05 for both). Feed efficiency was increased in Adcy9 Gt/Gt compared to WT mice ( P <0.05), which was accompanied by improved nocturnal heart rate variability ( P =0.0572) and prolonged cardiac RR interval ( P <0.05). Adcy9 inactivation-induced effects on atherosclerosis, endothelium-dependent vasodilation, weight gain and feed efficiency were lost in CETP Gt mice ( P >0.05 vs CETP WT ). Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, improved endothelial function and autonomic tone.
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