Investigating the effects of hypoxia on the sphingolipid system and adiponectin in adipocytes and perivascular adipose tissue

Almarshad, Saleh Ali Saleh (2026) Investigating the effects of hypoxia on the sphingolipid system and adiponectin in adipocytes and perivascular adipose tissue. PhD thesis, University of Glasgow.

Abstract

Perivascular adipose tissue (PVAT) exerts important paracrine control over vascular tone, but how acute hypoxia impacts upon PVAT signalling and vascular reactivity remains incompletely understood. Hypoxia is central to adipose pathophysiology because as the volume of adipose depots expand, limited oxygen diffusion and inadequate angiogenesis create localised hypoxia that drives metabolic stress, inflammation, and altered secretion of adipokines and bioactive lipid mediators. Together with impaired perfusion responses in obesity, these changes indicate that the regulatory role of PVAT may change and hypoxia may be central to this. To address this, I combined wire myography with molecular and biochemical measurements in rat thoracic aorta and mesenteric arteries, 3T3-L1 adipocytes, and ex vivo PVAT depots exposed to normoxia and hypoxia.

Functionally, pre-exposure to hypoxia altered subsequent contractile responses to phenylephrine and relaxant responses to cromakalim in a manner dependent on vessel type, PVAT status, and endothelium; when hypoxia was applied acutely during sustained phenylephrine-induced tone, thoracic aorta relaxed robustly even without endothelium, and relaxation was significantly augmented by PVAT, supporting an endothelium-independent component mediated by PVAT-derived factors.

Adiponectin is an abundant adipocyte-derived hormone with insulin-sensitising and anti-inflammatory actions and has recognised roles in vascular homeostasis. Therefore, adiponectin content and release were measured in 3T3-L1 adipocytes and in thoracic and mesenteric PVAT to determine if acute hypoxia alters PVATderived adiponectin and whether these changes could account for hypoxiainduced vascular relaxation. Adiponectin studies showed that short-term hypoxia reduced adiponectin protein and Adipoq mRNA in 3T3-L1 adipocytes and lowered adiponectin in thoracic but not mesenteric PVAT; under normoxic conditions, thoracic PVAT exhibited higher basal adiponectin protein content than mesenteric PVAT, and a trend toward increased release during hypoxia. Pharmacological manipulation revealed that, with PVAT present, neither β3-adrenoceptor agonist nor AdipoR1 blockade altered hypoxic relaxation; in PVAT-removed endotheliumintact rings, β3-agonism (CL-316,243) attenuated relaxation, indicating an inhibitory endothelial β3 pathway unmasked by PVAT removal. An exploratory adipokine array indicated broader hypoxia-induced reductions in several factors secreted from thoracic PVAT.

Sphingosine-1-phosphate is a PVAT-derived bioactive lipid produced by SphK1 that regulates vascular tone and plays key roles in cardiovascular and immune function. Hence, after observing that acute hypoxia induces relaxation with PVAT present, S1P was investigated as a candidate mediator linking hypoxia to vessel responses. SphK/S1P experiments demonstrated that hypoxia selectively and transiently increased SphK1 mRNA and SphK1 phosphorylation in adipocytes, while a short exposure to gas hypoxia did not change SphK1 protein or tissue S1P in PVAT. Thoracic PVAT released less S1P under hypoxia. Inhibition of SphK1 with PF543 reduced S1P release under normoxia and further decreased it under hypoxia, consistent with SphK1-driven S1P export from PVAT. Conversely, mesenteric PVAT showed no effect of PF543 or hypoxia on SphK1 or S1P release. Thus, the hypoxic reduction in S1P release was depot specific. Exogenous S1P or selective S1P2 activation (CYM 5478) reduced hypoxic relaxation only when PVAT was absent, suggesting that S1P/S1P2 can limit relaxation of the vessel wall but this is masked by PVAT factors when PVAT is present.

Collectively, these data show that acute hypoxia induces a PVAT-enhanced, largely endothelium-independent vasorelaxation while rapidly altering adiponectin and S1P signals. When PVAT is present, adiponectin and β3 agonism are not the dominant acute mediators of hypoxic relaxation, and S1P/S1P2 opposes relaxation primarily when PVAT support is removed. These findings refine the mechanistic understanding of PVAT to vessel crosstalk under hypoxic stress and point to PVAT factors as key acute effectors.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Supported by funding from Qassim University.
• Subjects: R Medicine > R Medicine (General); R Medicine > RC Internal medicine
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Supervisor's Name: Kennedy, Professor Simon and Watterson, Dr. Kenneth
• Date of Award: 2026
• Depositing User: Theses Team
• Unique ID: glathesis:2026-85700
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 22 Jan 2026 12:17
• Last Modified: 25 Jan 2026 09:05
• Thesis DOI: 10.5525/gla.thesis.85700
• URI: https://theses.gla.ac.uk/id/eprint/85700