title: The role of connexin 43 in the function of systemic and pulmonary blood vessels
Creators: name: Wali, Saad
Publisher: name: University of Glasgow
Year: 2024

description: Background: Connexins are membrane channel-forming proteins that play a pivotal role in direct intercellular communication within the vasculature. They are crucial in mediating various aspects of vascular physiology such as vasodilation and vasoconstriction, and also play a significant role in vascular pathology. The aim of this research is to elucidate the roles of connexins, particularly connexin 43 (Cx43), in the regulation of systemic and pulmonary circulations. The hypothesis is that Cx43 critically influences vascular reactivity and remodelling, which is particularly evident in pathological conditions like pulmonary hypertension. By investigating these roles, this study seeks to deepen our understanding of Cx43’s multifaceted functions in both physiological and pathophysiological contexts, including its involvement in disorders such as atherosclerosis, systemic hypertension, and pulmonary hypertension (PH).

Introduction: Connexin 43 (Cx43) is the most abundant isoform within the vasculature, and this thesis will examine its role in regulation of both the systemic and pulmonary circulation. In chapter 3 and 4, the role of Cx43 will be investigated in the systemic and pulmonary circulations respectively using mouse thoracic aortae, mouse intra-lobar pulmonary arteries (IPAs) and lungs. Pulmonary hypertension is a severe condition characterised by both pulmonary vascular remodelling and abnormal vasoconstriction, resulting in increased pulmonary vascular resistance and eventually leading to right-sided heart failure and death. Given the significant contribution of pulmonary artery fibroblasts (PAFs) to pulmonary vascular remodelling, chapter 5 will focus on examining the role of connexin 43 in proliferation and migration of mouse PAFs (MPAFs).

Methods: Thoracic aortae from 4- to 6-month-old mice and lungs and IPAs from 2- to 3-month-old mice were dissected free from age-matched female and male wild-type (WT) mice and connexin 43 heterozygous (Cx43+/−) mice from a C57BL/6 background. Using pentobarbital sodium (60mg/kg) and lidocaine (4mg/kg), mice were euthanized by intraperitoneal injection (i.p.). In all experiments, the role of Cx43 in the systemic and pulmonary vasculature was investigated using both pharmacological and genetic approaches. The gap junction blocker 37,43Gap27 was employed to pharmacologically inhibit Cx43 function. Additionally, Cx43+/− mice were used, wherein Cx43 expression is genetically reduced, not completely inhibited, providing a model to understand the impact of lower Cx43 levels on vascular function. The use of Cx43−/− mice, which completely lack Cx43, was not feasible due to lethal developmental defects that these mice experience, making them unsuitable for such studies. In the first part of this study, wire myography was used to assess both systemic and pulmonary vascular reactivity of thoracic aortae and IPAs. NO production was then investigated in whole thoracic aortae and lung tissues using a Sievers 280 analyser. Western blot was also used to investigate the protein expression of Cx43 and eNOS in the thoracic aortae and lungs. Cell culture was then used to examine the role of Cx43 in MPAFs. MPAFs were explanted from main and branch pulmonary arteries and were exposed to normoxic or hypoxic (5% O2) conditions for 24h. Proliferation and migration were assessed using an automated cell counter or a scratch assay respectively.

Results: Pharmacological inhibition and genetic reduction of Cx43 had effects on vascular reactivity and NO production in both the systemic and pulmonary circulations. In all of the experiments presented in this thesis on systemic and pulmonary vasoreactivity and NO assays, no significant differences between sexes were found. This study found that Cx43 associates with endothelial nitric oxide synthase and exists in the same protein complexes within whole thoracic aortae and lungs from both genotypes. This study further revealed that Cx43 significantly promotes the proliferation and migration of MPAFs under hypoxic conditions. This could ultimately lead to the remodelling of the pulmonary vasculature and subsequently leading to pulmonary hypertension.

Conclusion: This study has shown that Cx43 plays a role in systemic and pulmonary vasoreactivity and interacts with NO signalling pathways, indicating its potential as a pharmacological target. It is further demonstrated in this study that Cx43 is involved in hypoxic-induced cell proliferation and migration. Thus, Cx43 may be involved in the pulmonary vascular remodelling in response to hypoxia. This cellular process is of significant interest in the context of pulmonary vascular changes, and these findings should be seen as a foundation for further understanding the complex role of Cx43 in conditions like PH. Future research should investigate deeper into elucidating the specific molecular pathways involved and assessing the implications of targeting Cx43 therapeutically.