Régulation du cycle cellulaire par le récepteur natriurétique de type C dans les cellules du muscle lisse vasculaire : mécanismes moléculaires

Nous avons précédemment montré que l’activation du récepteur natriurétique de type C (NPR-C) par son agoniste spécifique, le C-ANP4-23, atténue l’augmentation de la prolifération des cellules du muscle lisse vasculaire (CMLV) induite par les peptides vasoactifs (Ang II, ET-1 et l’AVP). Puisque les CMLV provenant de rats spontanément hypertendus (SHR) montrent elles aussi un taux de prolifération plus élevé que leur contrôle, les CMLV de rats Wystar-Kyoto (WKY), nous avons entrepris cette étude dans le but de déterminer si C-ANP4-23 peut également diminuer le taux élevé de prolifération des CMLV de SHR et, le cas échéant déterminer les mécanismes responsables de cette réponse. Nos résultats montrent que le taux de prolifération des CMLV de SHR est significativement plus élevé que celui des CMLV de WKY et que la présence de C-ANP4-23 diminue de manière-dose dépendante le taux de prolifération des CMLV de SHR. En plus, l’expression des protéines de la phase G1 du cycle cellulaire, la cycline D1, la kinase dépendante des cyclines 2 (cdk2) et la forme phosphorylée de la protéine du rétinoblastome (pRb) est augmentée dans les CMLV de SHR comparativement aux CMLV de WKY et est atténué par C-ANP4-23. De plus, nos résultats montrent que les inhibiteurs du complexe cycline D1/cdk4 (NSC 625987) et cdk2 (NU2058) diminue le taux de prolifération élevé des CMLV de SHR. Les CMLV de SHR montrent également un taux de phosphorylation de ERK1/2 et d’AKT et est atténué par C-ANP4-23. De plus, le taux d’expression élevé des protéines cycline D1, cdk2 et pRb des CMLV de SHR est diminué par la toxine pertussis qui inactive la protéine Giα, le PD 98095, un inhibiteur de MEK de la voie des MAPK, du wortmannin, un inhibiteur de la PI3-K et finalement du losartan, un antagoniste du récepteur AT1. Ces résultats suggèrent que l’activation du récepteur NPR-C par C-ANP4-23 diminue le taux de prolifération élevé des CMLV de SHR par une régulation à la baisse des composantes du cycle cellulaire via l’inhibition de la protéine Giα et des voies signalétique MAP kinase/PI3-K.We have previously shown that natriuretic peptide receptor-C (NPR-C) activation by C-ANP4-23 decreased the proliferation of vascular smooth muscle cells (VSMC) induced by vasoactive peptides (Ang II, ET-1 and AVP). Since, VSMC from SHR also exhibit an enhanced proliferation as compared to VSMC from WKY, we undertook the present study to investigate if C-ANP4-23 could also attenuate the enhanced proliferation of VSMC from SHR and to further explore the underlying mechanisms responsible for this response. The proliferation of VSMC from SHR was significantly increased as compared to VSMC from WKY as determined by [3H]thymidine incorporation and was attenuated by C-ANP4-23 in a concentration-dependent manner. Furthermore the expression of cyclin D1, cyclin-dependent kinase 2 (cdk2) and phosphorylated retinoblastoma protein (pRb) was enhanced in VSMC from SHR compared to WKY which was attenuated by C-ANP4-23. In addition, the inhibitor of cdk4/cyclinD1 (NSC 625987) and cdk2 (NU2058) also attenuated the enhanced proliferation of VSMC from SHR in a concentration-dependent manner. VSMC from SHR also exhibited the enhanced phosphorylation of ERK1/2 and AKT as compared to WKY which was attenuated by C-ANP4-23.. Furthermore, the enhanced expression of cyclin D1, cdk2 and pRb in VSMC from SHR were also attenuated by pertussis toxin that inactivates Giα protein, PD 98095, a MEK kinase inhibitor, wortmannin, PI3K inhibitor as well as by losartan, an AT1 receptor antagonist. These results suggest that NPR-C activation attenuates the enhanced proliferation of VSMC from SHR which may be attributed to Giα /MAP kinase/PI3K-mediated inhibition of the expression of cell cycle components

Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated

The biological impact of blood pressure-associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle.

Elevated blood pressure (BP) is a major global risk factor for cardiovascular disease. Genome-wide association studies have identified several genetic variants at the NPR3 locus associated with BP, but the functional impact of these variants remains to be determined. Here we confirmed, by a genome-wide association study within UK Biobank, the existence of two independent BP-related signals within NPR3 locus. Using human primary vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) from different individuals, we found that the BP-elevating alleles within one linkage disequilibrium block identified by the sentinel variant rs1173771 was associated with lower endogenous NPR3 mRNA and protein levels in VSMCs, together with reduced levels in open chromatin and nuclear protein binding. The BP-elevating alleles also increased VSMC proliferation, angiotensin II-induced calcium flux and cell contraction. However, an analogous genotype-dependent association was not observed in vascular ECs. Our study identifies novel, putative mechanisms for BP-associated variants at the NPR3 locus to elevate BP, further strengthening the case for targeting NPR-C as a therapeutic approach for hypertension and cardiovascular disease prevention

Effect of C-ANP_4–23 on the expression of phosphorylated retinoblastoma protein (pRb) and retinoblastoma protein (Rb) in vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated in the absence (control) or presence 10^-7M of C-ANP_4–23 for 16 h. The cell lysates were prepared and used for Western blotting using specific antibodies against pRb (A) and Rb (B) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL taken as 100%. Values are means ± SE of 5 separate experiments. **<i>P</i><0.01.</p

Effect of in vivo treatment of C-ANP_4–23 on DNA synthesis in VSMC from 8 week-old SHR and age-matched WKY rats.

<p>One week old SHR and age matched WKY rats (control) were injected intraperitoneally with C-ANP_4–23 (10 nmol/Kg of body weight) twice weekly up to 8 weeks as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. After eight weeks of treatment, the rats were sacrificed and aortic VSMC from SHR and age-matched WKY (control groups) and C-ANP-_4–23-treated groups were cultured and thymidine incorporation was determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SE of 3 separate experiments.. *** P<0.001 vs WKY, ^### P<0.001 vs SHR.</p

Effect of C-ANP_4–23 on phosphorylation of ERK (A) and AKT(B) expression in vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated in the absence (control) or presence of different concentrations of C-ANP_4–23 for 16 h. The cell lysates were prepared and used for Western blotting using specific antibodies against pERK1/2 (A) and pAKT (B) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL taken as 100%. Values are means ± SE of 5 separate experiments. *<i>P</i><0.05, **<i>P</i><0.01.</p

Effect of C-ANP_4–23, NSC 625987and NU2058 on proliferation of Vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated in the absence (control) or presence of various concentrations of C-ANP_4–23 (A), NSC 625987 (B) or NU2058 (C) for 16 h. Thymidine incorporation was determined as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SE of 5 separate experiments. *** <i>P</i><0.01, ***<i>P</i><0.001 vs WKY CTL, #<i>P</i><0.05, ##<i>P</i><0.01 vs SHR CTL.</p

Effect of wortmannin on expression of cell cycle components from G1-phase in vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated for 16 h in the absence (control) or presence of 1 µM wortmannin WM). The cell lysates were prepared and used for Western blotting using specific antibodies against cyclin D1 (A), cdk4 (B), cdk2 (C) and pRB (D) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL taken as 100%. Values are means ± SE of 5 separate experiments. *<i>P</i><0.05, **<i>P</i><0.01, * **<i>P</i><0.001.</p

Effect of of C-ANP_4–23 on the expression of cell cycle components in vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated in the absence (control) or presence of C-ANP_4–23 (10⁻⁷M) for 16 h. The cell lysates were prepared and used for Western blotting using specific antibodies against cyclin D1 (A), cdk4 (B), cyclin A (C), cyclin E (D) and cdk2 (E) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL taken as 100%. Values are means ± SE of 5 separate experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Effect of C-ANP_4–23 on the expression of Giα-2 and Giα-3 proteins in vascular smooth muscle cells (VSMC) from SHR and WKY rats.

<p>VSMC from SHR and WKY rats were incubated in the absence (control) or presence 10^-7M of C-ANP_4–23 for 16 h. The cell lysates were prepared and used for Western blotting using specific antibodies against Giα-2 (A) and Giα-3 (B) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076183#s2" target="_blank">Methods</a>”. Results are expressed as % of WKY CTL, taken as 100%. Values are means ± SE of 5 separate experiments. **<i>P</i><0.01, ***<i>P</i><0.001.</p