Date of Award

5-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Kafait U. Malik, PhD

Committee

Modar O. Kassan, PhD; Edward A. Park, PhD; Ishrat Tauheed, PhD; Fuming Zhou, PhD

Keywords

12/15-Lipoxygenase, 17Beta-Estradiol, Angiotensin II, Hypertension, Kidney pathogenesis, Ovariectomy

Abstract

Background: Angiotensin (Ang) II releases arachidonic acid (AA) from tissue phospholipids that is metabolized by 12/15-lipoxygenase (ALOX15) generating 12(S)- and 15(S)-hydroxyeicosatetraenoic acid (HETE) which have been implicated in cardiovascular and renal diseases. This study was conducted to investigate a) the contribution of ALOX15 to Ang II-induced hypertension and associated pathogenesis in female mice, and b) the interaction between the cytochrome P450 (CYP)1B1-generated metabolite of 17β-estradiol (E2) (2-methoxyestradiol, 2-ME) and ALOX15 in the paraventricular nucleus (PVN) as a mechanism that protects against Ang II-induced hypertension and its associated pathogenesis in female mice. Methods: Experiments were conducted in intact and ovariectomized (OVX) wild type (WT), alox15 knockout (ALOX15KO) and CYP1B1KO female mice. Ang II (700 ng/kg/min) was infused subcutaneously by osmotic pumps for 2 weeks evaluation of hypertension and associated pathogenesis. Blood pressure (BP) was measured by tail-cuff and confirmed by radiotelemetry. Adenoviral probes including adenovirus (Ad)-green fluorescence (GFP)-ALOX15-short hairpin (sh)RNA, Ad-GFP-ALOX15-DNA, and their respective controls Ad-scrambled shRNA and Ad-GFP-DNA, 12(S)-HETE and 2-ME were injected in the brain using stereotaxic technique, either selectively in PVN or intracerebroventricularly via ICV cannula implanted in the right lateral ventricle to study their roles on Ang II-induced hypertension. Histological, immunohistochemical, and fluorescence microscopy and biochemical techniques were employed to determine the pathophysiological changes in tissues. ELISA was used for the analysis of sex steroids and eicosanoids. Results: Our results demonstrate that the effects of Ang II to increase BP, impair autonomic function and increase renal reactive oxygen species (ROS) production and plasma 12(S)-HETE but not 15(S)-HETE level without altering renal function in intact WT mice were exacerbated in OVX-WT mice. Ang II also increased renal alox15 mRNA, urine 12(S)-HETE, water intake, urine output, decreased osmolality, increased urinary excretion of vasopressin prosegment copeptin, protein/creatinine ratio, and caused renal hypertrophy, fibrosis, and inflammation in OVX-WT mice. These effects of Ang II were attenuated in ALOX15KO mice. Moreover, the Ang II-induced hypertension that was also exaggerated in intact CYP1B1KO mice compared to WT mice was minimized by selective alox15 gene knockdown in PVN by transduction with Ad-ALOX15-shRNA and the restoration of Ang II-induced hypertension by selective reconstitution of alox15 gene in PVN by transduction with Ad-ALOX15-DNA in intact ALOX15KO mice was further exacerbated in OVX-ALOX15KO mice. Furthermore, ICV-12(S)-HETE that restored Ang II-induced increase in BP, impairment of autonomic function, neuroinflammation and renal pathogenesis in intact ALOX15KO mice, further exacerbated these effects of Ang II in OVX-ALOX15KO mice. Finally, ICV-2-ME that reduced the alox15 mRNA expression and 12(S)-HETE content in PVN minimized the hypertensive effects of Ang II, including BP, autonomic impairment, neuroinflammation and renal pathogenesis in OVX-ALOX15KO with the restoration of alox15 gene in PVN by transduction ICV with Ad-ALOX15-DNA. Conclusion: These data suggest that 17β-estradiol plays a critical role in female mice in protecting against Ang II-induced hypertension and associated pathogenesis, most likely via inhibition of ALOX15 activation and reduced production of 12(S)-HETE in the PVN. Therefore, the selective inhibitors of ALOX15 or 12(S)-HETE receptor antagonists could be useful for treating hypertension and its pathogenesis in postmenopausal, hypoestrogenic women or females with ovarian failure. These data also elucidate how drugs that inhibit CYP1B1 activity can be beneficial for treating hypertension and its pathogenesis in males but can be detrimental in females. The effect of alox15 gene polymorphism in pre-and postmenopausal females to determine its impact on the contribution of AA-ALOX15 derived 12(S)-HETE in hypertension and its pathogenesis is also warranted.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0003-3643-1200

DOI

10.21007/etd.cghs.2023.0628

Additional Files
2023-015-Dutta-DOA.pdf (127 kB)
Declaration of Authorship
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