Differential regulation of vascular tone and remodeling via stimulation of type 2 and type 6 adenylyl cyclases in the ductus arteriosus

Abstract

Rationale: Prostaglandin (PG)E(2), which increases intracellular cAMP via activation of adenylyl cyclases (ACs), induces vasodilation and hyaluronan-mediated intimal thickening (IT) in the ductus arteriosus (DA) during late gestation. After birth, however, differential regulation of vasodilation and IT is preferable for treatment of patients with patent DA and DA-dependent congenital cardiac malformations.

Objective: Our objectives were to examine whether AC isoforms play differential roles in DA vasodilation and IT.

Methods and results: AC2 and AC6 were more highly expressed in rat DA than in the aorta during the perinatal period. AC6-targeted siRNA counteracted PGE(1)-induced hyaluronan production in rat DA smooth muscle cells. Overexpression of AC6 enhanced PGE(1)-induced hyaluronan production and induced IT in DA explants. Furthermore, IT of the DA was less marked in mice lacking AC6 than in wild-type and AC5-deficient mice. Stimulation of AC2 attenuated AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced IT of the DA. An AC2/6 activator, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1), did not induce hyaluronan-mediated IT in DA explants, although an AC5/6 activator, 6-[3-(dimethylamino)propionyl]-14,15-dihydroforskolin (FD6) did. Moreover, FD1 induced longer vasodilation of the DA than did PGE(1) without significant adverse effects in vivo.

Conclusions: AC6 is responsible for hyaluronan-mediated IT of the DA and AC2 inhibited AC6-induced hyaluronan production. Stimulation of both AC2 and AC6 by FD1 induced longer vasodilation without hyaluronan-mediated IT in the DA in vivo. FD1 may be a novel alternative therapy to currently available PGE therapy for patients with DA-dependent congenital heart disease.

Figure 1

Multiple transcripts of AC isoforms in rat DA. (A) mRNA expression of AC isoforms using semi-quantitative RT-PCR in rat e21 DA. (B) Quantitative RT-PCR analyses of AC2-7. **P<0.01. Data are from six independent experiments.

Figure 2

AC6 is responsible for hyaluronan (HA) production in DASMCs. (A) PGE1 induced cAMP accumulation in DASMCs in the cells treated with negative siRNA. AC2-, AC5-and AC6-targeted siRNA decreased PGE1-induced cAMP production (n=4). (B) AC6-targeted siRNA attenuated PGE1-induced hyaluronan production, whereas AC2-and AC5-targeted siRNA did not (n=7-11). (C) Adv.AC6 enhanced PGE1-induced hyaluronan production. Adv.AC2 abolished the Adv.AC6-induced enhancement of hyaluronan production (n=4). *P<0.05 and ***P<0.001. NS: not significant.

Figure 3

Adenovirus-mediated AC6 gene transfer promoted IT in rat DA explants. (A) Elastica van Gieson staining for cultured DA explants overexpressed with Adv.LacZ, Adv.AC2, Adv.AC6 or Adv.AC2+Adv.AC6. (B) Strong immunoreaction to hyaluronan in DA explants cultured with Adv.AC6. Bars=100 μm. (C) The area of the internal lumen of the DA treated with Adv.AC6 was significantly decreased (n=8-9). (D) The ratio of IT to the thickness of the medial layer was increased in the DA treated with Adv.AC6, but not with Adv.AC2 (n=8-9). ***P<0.001. HABP: hyaluronan binding protein.

Figure 4

Impaired IT in the mouse DA due to AC6, but not AC5, deficiency. (A and C) DAs from AC5KO mice at e18.5 were stained with Elastica van Gieson stain. Both AC5KO and WT mice showed IT in the DA (n=4-5). (B and D) DAs from AC6KO mice at e18.5 had less IT compared to WT mice (n=8). Bars=50 mm. ***P<0.001.

Figure 5

The effects of FD1 and FD6 on cAMP and hyaluronan production in DASMCs. (A) the effect of overexpression of AC2 or AC6 on FD1- or FD6-induced cAMP accumulation (n=6). (B) FD1 and FD6 increased cAMP accumulation in DASMCs in a dose-dependent manner (n=4). (C) FD6, but not FD1, increased hyaluronan production (n=8-11). (D) FD6 significantly increased HAS2 mRNA (n=6). (E) AC6-targeted siRNA negated FD6-induced hyaluronan production (n=4). (F) FD6, PGE1 and forskolin did not induce hyaluronan production in aortic SMCs. (n=6) **P<0.01 and ***P<0.001 vs. CTRL. Fsk: forskolin.

Figure 6

The signaling pathway of AC6-induced hyaluronan production in DASMC. (A) Phosphorylation of p38 protein (p-p38) by FD1 (10-5.5mol/L), FD6 (10-5mol/L) or Bnz-cAMP (10-5mol/L) (n=4). (B) Quantification of the ratio of p-p38 to total p38 after 5 min stimulation by FD1, FD6 or Bnz-cAMP. (n=4) (C) Phosphorylation of p38 and MKK3/6 induced by 5 min treatment of FD1 (10-5.5mol/L) or FD6 (10-5mol/L) in DASMCs treated with si-negative, si-AC2 or si-AC6 RNA (n=4). (D) FD6-induced hyaluronan production was attenuated by SB203580 (SB, 2X10-5mol/L) and H89 (10-6mol/L). (n=6) (E) SB203580 (2x10-5mol/L) did not affect Bnz-cAMP-induced hyaluronan production. (n=6) (F) MKK3 protein expression by Adv.MKK3. (G) Adv.MKK3 enhanced FD6-induced hyaluronan production. (n=6) *P<0.05, **P<0.01 and ***P<0.001.

Figure 7

IT in rat DA explants is promoted by FD6. (A) Elastica van Gieson staining for DA explants treated with FD1 or FD6. (B) Strong immunoreaction to hyaluronan in DA explants cultured with FD6. (C) The area of the internal lumen of the DA treated with FD6 was significantly decreased (n=6-7). (D) The increased IT in the DA treated with FD6 (n=6-7). ***P<0.001 vs. control (CTRL). Bars=100 μm. HABP: hyaluronan binding protein.

Figure 8

The effects of FD1 and FD6 on vasodilation. (A) FD1 and FD6 similarly attenuated indomethacin-induced contraction in DA explants in a dose-dependent manner (n=4-10). (B) The whole body freezing method revealed that the rat DA opens widely after birth and closed 1 h after birth (arrow, n=5). (C) Representative images of rat DAs treated with PGE₁ and FD1 using the whole body freezing method. (D) The vasodilating effects of PGE₁, FD1 or FD6 were compared (n=5-7). FD1 had a longer action of duration than did PGE₁. All rats injected with FD6 died 4h after injection due to apnea. Bars=1 mm. PA: pulmonary artery