The Biology of Kruppel-Like Factor 14 in Macrophages and Its Translational Potential in Cardiovascular Diseases

Abstract

Krüppel-like factor 14 (KLF14) is a transcription factor that belongs to the Krüppel-like factor family, containing three zinc finger domains on the C-terminal that directly binds DNA and regulates target gene transcription. Single nucleotide polymorphisms (SNPs) proximal to KLF14 are associated with lower KLF14 expression and higher risks of cardiovascular diseases (CVDs). However, the biological functions and mechanisms of KLF14 in protecting against CVDs remain largely unknown. Macrophages are involved in various CVD pathogenesis, including atherosclerosis and abdominal aortic aneurysm (AAA), characterized by excessive local lipid accumulation and higher chronic inflammation. Under pro-atherogenic conditions, KLF14 expression is significantly reduced in macrophages as well as in mice aortas. The first part of this study showed that KLF14 overexpression could promote cholesterol efflux and inhibit pro-inflammatory response in THP-1-derived macrophages by upregulating ATP-binding cassette transporter A1 (ABCA1) and inhibit-ing Interleukin-1β (IL-1β) expression. On the other hand, Klf14 deficiency reduced cholesterol efflux, increased lipid accumulation, and increased pro-inflammatory response in primary macrophages. Indeed, myeloid-specific Klf14 knockout mice fed Western Diet for 12 weeks showed aggravated atherosclerosis with higher macrophage infiltration and cholesterol crystal formation in the atheroma. Perhexline, an anti-anginal drug, was screened as a KLF14 inducer that directly up-regulates KLF14 expression. Administration of perhexiline up-regulates ABCA1 in a Klf14-dependent manner and inhibits IL-1β expression in macrophages without inducing hepatic lipogenesis, implicating KLF14 as a potential therapeutic target for treating or preventing atherosclerosis. Abdominal aortic aneurysm (AAA) is a disease showing both high prevalence and high mortality in aged populations without any effective non-surgical treatment. Male gender is a strong risk factor for AAA, with a 4-6:1 male to female prevalence ratio in the human population. Similar sex differences could also be observed in several AAA animal models. The endogenous protective effect observed in female individuals during AAA pathogenesis is primarily attributed to estrogen/estrogen receptor (ER) signaling. The second part of my thesis work revealed that myeloid-specific Klf14 deficiency ablated the sex differences observed between male and female mice. Compared to female wild-type mice, female myeloid-specific Klf14 knockout mice showed a significantly higher AAA incidence rate, with more macrophage infiltration and increased elastin degradation, to an extent similar to those in male wild-type mice. Furthermore, myeloid Klf14 deficiency completely abolished the exogenous E2-mediated protective effects in the Angiotensin II (Ang II) infusion-induced AAA mouse model. Mechanistically, KLF14 overexpression could significantly up-regulate the mRNA and protein levels of estrogen receptor β (ERβ, encoded by ESR2) in macrophages via binding to the promoter of the human ESR2 gene and regulating its transcription. Perhexiline treatment could induce Esr2 expression in a Klf14-dependent manner in macrophages. Induction of KLF14 by perhexiline inhibited the expression of IL-1β, while this effect was partially blocked in Esr2-deficiency peritoneal macrophages. Moreover, administration of perhexiline significantly improved the survival rate of mice during AAA progression, suggesting that KLF14 is also a potential therapeutic target for AAA treatment. This study revealed that KLF14 protects against AAA development via ER-mediated anti-inflammatory effect. In summary, my thesis work demonstrate that KLF14 is a promising therapeutic target to protect against or treat atherosclerosis and AAA. It does so by inducing cholesterol efflux, inhibiting inflammatory responses, and reducing MMP2/9 activities in macrophages. In addition, myeloid KLF14 also mediates sexual dimorphism in AAA development via upregulating ERβ expression.