Assessing adenoviral delivery of prolylcarboxypeptidase and extracellular vesicle-mediated delivery of angiotensin converting enzyme 2 as therapies in hypertensive heart disease

Alhajoj, Ahmad Mohammed A. (2024) Assessing adenoviral delivery of prolylcarboxypeptidase and extracellular vesicle-mediated delivery of angiotensin converting enzyme 2 as therapies in hypertensive heart disease. PhD thesis, University of Glasgow.

Abstract

Cardiovascular diseases (CVD) are group of disorders that involve the heart and blood vessels and are considered the main cause of death globally. Chronic activation of the classical renin angiotensin system (RAS) via angiotensin converting enzyme (ACE)/Angiotensin II (Ang II) type 1 receptor (AT1R) is associated with CVD including cardiac hypertrophy, and inhibition of this signalling pathway via ACE inhibitors or AT1R blockers (ARBs) can reduce or inhibit cardiac remodelling. Another mechanism to protect the heart from Ang IIdependent remodelling is via stimulation of the counter-regulatory axis of the RAS, mainly angiotensin converting enzyme 2 (ACE2)/angiotensin-(1-7) Ang-(1- 7)/Mas receptor signalling pathway. Prolylcarboxypeptidase (PRCP) is an alternative enzyme contributing to the counter-regulatory axis of the RAS via converting Ang II into the protective peptide Ang-(1-7). However, less attention has been given to PRCP’s role in CVD. The aims of this thesis were to generate an adenoviral (Ad) gene transfer vector expressing rat PRCP (Ad-PRCP), and to load EVs-with ACE2 (ACE2 EV) and study their therapeutic effects on angiotensin peptides metabolism and cardiac hypertrophy in vitro and in vivo. Successful generation of Ad-PRCP was confirmed by sequencing and PRCP gene expression in vitro. Next, Ang II was added to HeLa cells and levels measured at specific time points following transduction with Ad-PRCP or control Ad-GFP. Reduced Ang II levels were observed in Ad-PRCP transduced HeLa cells after 24hr of Ang II stimulation. In H9c2 cardiomyocytes, Ang II levels were significantly higher in Ang II only treated and in Ad-GFP+Ang II groups compared to control. Ad-PRCP+Ang II did not significantly change at 24h from either control or other Ang II stimulated groups. Furthermore, 48hr Ang II stimulation significantly increased Ang-(1-7) levels in Ad-PRCP+Ang II, but not Ad-GFP+Ang II transduced cardiomyocytes, compared to control untreated cells. Assessment of cardiomyocyte hypertrophy following Ang II stimulation revealed that cell surface area was significantly increased in comparison to control, an effect also observed following Ad-GFP+Ang II, but not in Ad-PRCP+Ang II treated groups. These data suggested that overexpression of PRCP might diminish Ang II- 18 mediated cardiomyocyte hypertrophy in vitro. Next, the protective effect of AdPRCP following 3 weeks of Ang II infusion in Wistar Kyoto rats (WKY) was investigated. Ad-PRCP showed no effect on Ang-II-dependent hypertension or cardiac remodelling. Analysis of cardiac function showed no significant changes in ejection fraction (EF%) and fractional shortening (FS%) within a group over time. However, after 3 weeks EF% and FS% were significantly reduced in Ang II group compared to Ad-PRCP+Ang II group. No other significant changes in cardiac function were observed between groups at 3 week time point. The potential to load EVs with ACE2 and their therapeutic effects on Ang IIdependent H9c2 cardiomyocyte hypertrophy was assessed. EVs were isolated from media of H9c2 cardiomyocytes transduced with Ad-ACE2 (ACE2 EV), Ad-GFP (GFP EV) or untransduced (control EV). The concentration of ACE2 EV was significantly greater than control EV and GFP EV. The average mean size for all EV groups was less than 200 nm. EV-associated markers, CD63 and TSG101, were detected in ACE2 EV and GFP EV via western blot, while ACE2 was observed only in ACE2 EV. Furthermore, ACE2 protein expression was detected in the cell lysate of H9c2 cardiomyocytes treated with ACE2 EV, but not in control or GFP EV treated groups. This observation suggested ACE2 could be loaded into EVs and is detectable via western immunoblotting. Next, ACE2 activity in each EV group was assessed revealing significantly higher ACE2 enzyme activity only in ACE2 EV group. Ang II and Ang-(1-7) levels were measured in control untreated cells or cells stimulated with Ang II alone or Ang II with either ACE2 EV or GFP EV. No statistically significant changes in Ang II or Ang-(1-7) were observed. The protective effects of ACE2 EV against Ang II-mediated cardiomyocyte hypertrophy were investigated. ACE2 EV diminished Ang II-induced H9c2 cardiomyocyte hypertrophy. These data suggested ACE2 EVs might be protective against Ang II-dependent H9c2 cardiomyocyte hypertrophy. In conclusion, the delivery of both enzymes, PRCP via adenoviral gene transfer vector and ACE2 via extracellular vesicles, exhibited potential therapeutic effects against Ang II-induced cardiac remodelling. Further studies are required to fully investigate each therapeutic approach in CVDs.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > R Medicine (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Nicklin, Professor Stuart and Loughrey, Professor Christopher
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84288
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 01 May 2024 14:53
• Last Modified: 01 May 2024 14:56
• Thesis DOI: 10.5525/gla.thesis.84288
• URI: https://theses.gla.ac.uk/id/eprint/84288