A synthetic angiotensin II/ACE2-based hormone shunt controlling experimental hypertension

Figures

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  Fig. 1. Establishing an Ang-II-dependent hypertension sensor in mammalian cells. a The 8-amino-acid peptide hormone Ang-II endogenously interacts with the receptor AT1R within the renin angiotensin system (RAS), leading to elevation of blood pressure. In our designer cell circuit, we rewire this interaction to trigger the expression of a reporter gene. b The sensor circuit was implemented in a variety of rodent (BHK-21, NIH/3T3), canine (MDCK), primate (COS-7), and human (HEK-293T, hMSC, HepG2) cells. Signaling was functional in all cell lines examined, but optimal transcriptional control was achieved with HEK-293T cells. c Various synthetic promoters were tested with AT1R (pGU52). The engineered promoter CSN containing triple sequence repeats of SRE, NFAT, and CRE (pGU23, PCSN-SS-nLuc-Fc-pA) produced the highest expression level as well as the tightest control. In (b, c): data shown as mean ± SD, n = 3 biological replicates, statistical analyses represent unpaired, two-tailed Welch’s t-test with a 95% confidence interval, where ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data for this figure is available in the Source data file.

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  Fig. 2. Optimization of the ARCH sensor platform in human cells. a Schematic of the renin-angiotensin system (RAS). The RAS contains various angiotensin analogs that interact with different GPCRs. Ang-II is converted by aminopeptidase A (APA) to Ang-III or by ACE2 to Ang-(1–7). Ang-III is further cleaved by aminopeptidase N (APN) to Ang-IV, while Ang-(1–7) is converted to alamandine (Ala) by aspartate decarboxylase (AD). Ang-II and to a lesser extent Ang-III interact with AT1R, Ang-II also interacts with AT2R, whereas the other angiotensin analogs (Ang-IV, Ang-(1–7), Ala) are ligands to other GPCRs (AT4R, MasR, MRGD) within the RAS. b Specificity of AT1R activation. HEK-293T cells were transfected with ARCH sensor plasmids (pGU23, pGU52) and exposed to 10−6 M angiotensin analogs present in the RAS. Only Ang-II and Ang-III were capable of activating the receptor. The angiotensin analogs generated by the ACE2 enzymatic reaction were unable to activate ARCH. Data shown as mean ± SD, n = 3 biological replicates. c Receptor tunability. Increasing quantities of the transfected receptor vector (pGU52) at fixed reporter (30 ng pGU23) and total plasmid amounts show peak expression and…

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  Fig. 3. Generation of stable HEKARCH cells. a The binding of Ang-II to AT1R triggers activation of the broad-spectrum CSN promoter, which integrates the signaling to initiate therapeutic stACE2 expression. The stACE2 expressed by HEKARCH cleaves Ang-II into Ang-(1–7), rendering it unable to interact with AT1R, and thereby causing (i) the lowering of blood pressure and (ii) the slowing of the therapeutic circuit through feedback inhibition. b SEAP reporter expression and fold-induction levels of HEK-293T cells with transient CAG-driven human AT1R (pGU52, PCAG-hAT1R-pA) and CSN-driven SEAP expression (pYL1, PCSN-SEAP-pA), as well as of polyclonal cell populations stably expressing human AT1R with transient CSN-driven SEAP expression (pYL1, PCSN-SEAP-pA). c Stability of monoclonal HEKARCH circuit performance across 30 passages. d Ang-II dose-response of a polyclonal cell population with stably integrated PCAG-driven human AT1R (pGU52) and transient PCSN-driven SEAP expression (pYL1), compared with monoclonal HEKARCH cells with stably integrated PCAG-driven human AT1R (pGU52) and PCSN-driven stACE2 and SEAP (pGU139, PCSN-stACE2-IgG1-P2A-SEAP-pA) expression. e In vitro Ang-II dose-response…

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  Fig. 4. Interaction of ARCH with canonical hypertension medications. a One candidate from each of the five main classes of hypertension medication was tested with HEKARCH having stably integrated CAG-driven human AT1R (pGU52) and CSN-driven stACE2 and SEAP (pGU139, PCSN-stACE2-IgG1-P2A-SEAP-pA) expression. stACE2 release was blocked only by a working concentration of the clinically licensed AT1R antagonist valsartan, which interferes with ARCH’s Ang-II detection and signaling. All analyses are two-tailed t-tests versus the Ang-II-only group. b The competitive inhibitor valsartan and stACE2 were both able to inhibit the Ang-II/AT1R interaction with similar kinetics in vitro. Statistical comparison is column dependence from 2-way ANOVA. In (a, b), data shown as mean ± SD, n = 3 biological replicates. In (a, b): statistical analyses represent unpaired, two-tailed Welch’s t-test with a 95% confidence interval, where ns not significant; ***p < 0.001. Source data for this figure is available in the Source data file.

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  Fig. 5. ARCH for angiotensin-dependent hypertension. a Schematic of the in vivo experimental model depicting the cell-laden capsule implants as well as the tail-cuff device used to record blood pressure values non-invasively. b Blood pressure readings of hypertensive (HT) treated and untreated mice, as well as wild-type (WT) treated and untreated mice, were taken every morning for 10 days. Statistical comparison shown is between the groups [HT mice – HEKARCH] and [HT mice + HEKARCH]. c Single, high-dose intraperitoneal valsartan injection (25 mg/kg) lowered the blood pressure of hypertensive mice gradually over 3 days. d The disease group treated with ARCH showed a rapid improvement that remained stable for the entire duration of the study. ARCH had no apparent negative effects on the wild-type treated group. Each data point is an individual blood pressure reading starting from day 5. Plasma isolated from day 10 blood samples of each mouse was analyzed with a human ACE2 ELISA kit for stACE2 quantification (e) and with a luminescence reporter assay kit for SEAP quantification (f). Data shown as mean ± SEM, n = 4 mice per group. In (b, c): statistical analyses represent the linear mixed…

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  Fig. 6. ARCH for RAS dysregulation. a Plasma Ang-II levels gradually increased in obese mice, and the increase was attenuated by the HEKARCH implant. The ACE2-mediated therapeutic effect was transiently blocked by intraperitoneal bolus injections of ACE2 inhibitor (MLN4760, 1 mg/kg). b Renal Ang-II levels were significantly reduced in treatment groups. c Plasma ACE2, monitored twice weekly, was inversely correlated with plasma Ang-II attenuation. d No significant changes were observed in membrane-bound renal ACE2 levels. e Plasma Ang-(1–7) levels were transiently elevated in treatment groups following HEKARCH implantation but subsequently remained stable throughout the experiment. f No changes were observed in renal Ang-(1–7). g, h Plasma and renal renin were elevated in the [+HEKARCH − MLN4760] group. In (a, c, e, g): statistical comparison shown is between the groups [+HEKARCH − MLN4760] and [−HEKARCH − MLN4760]. The green arrow indicates the therapeutic cell implantation procedure on day 5, and the blue arrows indicate the intraperitoneal bolus injections of ACE2 inhibitor on day 14 and day 18 (MLN4760, 1 mg/kg). Data shown as mean ± SEM, n = 4 mice per group. In (a–h): statistical…

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  Fig. 7. ARCH response to human samples. a, b Blood pressure of patients who provided serum samples. Elevated systolic and normal diastolic blood pressure measurements indicate isolated systolic hypertension (ISH). c Plasma renin levels of the selected patients were two to five times higher than the cohort average. d Plasma angiotensin levels of the selected patients were in the 80th percentile. e Plasma Ang-II levels. f HEKARCH cultured in human samples achieved significant stACE2 expression for two of the four patients. In (c, d): solid line indicates the cohort mean, dashed line indicates the cohort median. In (e, f): statistical analyses represent unpaired, two-tailed Welch’s t-test with a 95% confidence interval, where n = 3 biological replicates; data shown as mean ± SEM. *p < 0.05, **p < 0.01. Source data for this figure is available in the Source data file.