Overexpression of Central ACE2 (Angiotensin-Converting Enzyme 2) Attenuates the Pressor Response to Chronic Central Infusion of Ang II (Angiotensin II): A Potential Role for Nrf2 (Nuclear Factor [Erythroid-Derived 2]-Like 2).

We investigated the mechanism by which ACE2 (angiotensin-converting enzyme 2) overexpression alters neurohumoral outflow and central oxidative stress. Nrf2 (nuclear factor [erythroid-derived 2]-like 2) is a master antioxidant transcription factor that regulates cytoprotective and antioxidant genes. We hypothesized that upregulation of central ACE2 inhibits the pressor response to Ang II (angiotensin II) by reducing reactive oxygen species through a Nrf2/antioxidant enzyme-mediated mechanism in the rostral ventrolateral medulla. Synapsin human Angiotensin Converting Enzyme 2 positive (SynhACE2+/+) mice and their littermate controls synhACE2-/- were used to evaluate the consequence of intracerebroventricular infusion of Ang II. In control mice, Ang II infusion evoked a significant increase in blood pressure and norepinephrine excretion, along with polydipsia and polyuria. The pressor effect of central Ang II was completely blocked in synhACE2+/+ mice. Polydipsia, norepinephrine excretion, and markers of oxidative stress in response to central Ang II were also reduced in synhACE2+/+ mice. The MasR (Mas receptor) agonist Ang 1-7 and blocker A779 had no effects on blood pressure. synhACE2+/+ mice showed enhanced expression of Nrf2 in the rostral ventrolateral medulla which was blunted following Ang II infusion. Ang II evoked nuclear translocation of Nrf2 in cultured Neuro 2A (N2A) cells. In synhACE2-/- mice, the central Ang II pressor response was attenuated by simultaneous intracerebroventricular infusion of the Nrf2 activator sulforaphane; blood pressure was enhanced by knockdown of Nrf2 in the rostral ventrolateral medulla in Nrf2 floxed (Nrf2f/f) mice. These data suggest that the hypertensive effects of intracerebroventricular Ang II are attenuated by selective overexpression of brain synhACE2 and may be mediated by Nrf2-upregulated antioxidant enzymes in the rostral ventrolateral medulla.

Upregulating Nrf2 in the RVLM ameliorates sympatho-excitation in mice with chronic heart failure.

Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that maintains redox homeostasis by governing a broad array of antioxidant genes in response to oxidant stress. We hypothesized that overexpression of Nrf2 in the rostral ventrolateral medulla (RVLM) ameliorates sympatho-excitation in mice with coronary artery ligation-induced chronic heart failure (CHF). To address this, we overexpressed Nrf2 in the RVLM using an HIV-CamKIIa-Nrf2 lenti virus in C57BL/6 mice. In addition, we used a Lenti-Cre virus in Keap1flox/flox mice to upregulate Nrf2 non-selectively in the RVLM. Arterial blood pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded under conscious and anesthetized conditions, respectively. Protein expression was assayed using western blotting and immunofluorescence staining. We found that (1) Nrf2 and two target proteins, NQO1 and HO-1 in the RVLM were significantly lower in CHF compared to Sham mice. Nrf2 viral transfection of the RVLM upregulated Nrf2 protein. (2) Urinary NE excretion in CHF mice was markedly attenuated following Nrf2 upregulation (812 ±â€¯133 vs 1120 ±â€¯271 ng/24hr mean. ±SE, *p < 0.05, n = 8/group). (3) In the conscious state, CHF mice overexpressing Nrf2 exhibited an enhancement in spontaneous baroreflex gain and in phenylephrine-induced baroreflex control of HR. (4) Acute experiments under anesthetisa revealed a significant decrease in basal RSNA (44.0 ± 6.5 vs 64.7 ± 8.3% of Max. *P < 0.05 n = 8/group) and enhancement in baroreflex sensitivity (Maximal gain -1.8 ± 0.3 vs 1.1 ± 0.2 of mmHg. **p < 0.01. n = 6/group) in CHF mice that were virally transfected with Nrf2 compared with CHF mice transfected with Lenti-GFP. Finally, Lenti-Cre viral overexpression of Nrf2 in Keap1flox/flox mice reduced Keap1 protein and increased Nrf2, NQO1, and HO-1 in the RVLM of Sham and CHF mice. CHF-Cre mice exhibited a significant decrease in baseline RSNA and plasma NE concentration (8.9 ± 1.1 vs 12.7 ± 0.9 ng/mL *P < 0.05 n = 6/group) as compared with CHF-GFP mice. Based on the above data, we conclude that upregulating Nrf2 selectively in the RVLM attenuates sympatho-excitation in CHF mice. Nrf2 may be an important central target for autonomic modulation in cardiovascular disease and during stress.

Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling.

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

MAZ mediates the cross-talk between CT-1 and NOTCH1 signaling during gliogenesis.

Neurons and glia cells are differentiated from neural stem/progenitor cells (NSCs/NPCs) during brain development. Concomitant activation of JAK/STAT and NOTCH1 signaling is required for gliogenesis, a process to generate glia cells to ensure proper brain functions. NOTCH1 signaling is down-regulated during neurogenesis and up-regulated during gliogenesis. However, the underlying mechanism remains elusive. We report here that cardiotrophin-1 (CT-1) activates NOTCH1 signaling through the up-regulation of ADAM10, a rate-limiting factor of NOTCH1 signaling activation. We found that a transcriptional factor, Myc-associated zinc finger protein (MAZ), plays an important role in ADAM10 transcription in response to CT-1 in NPCs. MAZ knockdown inhibits CT-1 stimulated gliogenesis and it can be rescued by over-expressing human NICD. Our results provide a link between NOTCH1 activation and neuronal secreted CT-1, suggesting that CT-1 plays an important role in ensuring the coordinated activation of NOTCH1 signaling during gliogenesis.