TITLE The novel mechanisms of HIV associated vascular cognitive impairment ABSTRACT The World Health Organization (WHO) reported there are approximately 38 million people living with HIV (PLWH) around the world at the end of 2019. In last two decades, the widespread deployment of combination antiretroviral therapy (cART) has led to a substantially decline of death rate in PLWH and increase of life expectancy. However, the aging HIV population is experiencing an increased burden of cerebrovascular disease greater than expected in the general population and HIV associated vascular cognative impairment (HIV-VCI), even vascular dementia (VD, a severe form of VCI) is one major type. The underlying mechanisms is still unknown.The goal of the current proposal is to determine the role of monocyte derived extracellular Delta like-4 (Dll4) in the progression of HIV- VCI. Dll4 is a Notch ligand and is usually restricted in endothelial cells (ECs) of small vessels in adult human and mouse brain. The ectopic expression of Dll4 in monocyte provides the link between systemic inflammation and brain vessel remodeling leading to HIV-VCI. In vitro, we demonstrated that LPS induced a robust increase of Dll4 mRNA and protein expression in human monocytes and Dll4 secretion from monocytes (extracellular Dll4, exDll4). In vivo, mice injected with exDll4 show cerebrovascular remodeling. In clinical specimen, we found that HIV patients on cART show a remarkable elevated plasma exDll4. The plasma exDll4 was positively associated with intermediate subpopulation of monocytes (pro-inflammatory monocytes), indicating a strong correlation of circulating exDll4 with inflammation. Importantly, circulating exDll4 was significantly associated with brain vessel diameter in HIV+ patients. In inflammatory mouse model induced by LPS, we observe remarkable enhanced Dll4 expression in monocytes and brain vascular remodeling.To explore the mechanisms of exDll4, we treated the human endothelial cells (ECs), and human smooth muscle cells (SMCs) with exDll4. Our vitro studies showed that exDll4 significantly inhibited Notch1 activation in ECs and repressed both Notch2 and Notch3 activation in SMCs. Decreased Notch1 activation in ECs leads to increase of EC permeability. Therefore, we hypothesize that monocyte derived Dll4 promotes the progression of VCI by impairing the blood brain barrier (BBB) and increasing brain small vessel remodeling through inhibiting Notch signaling in ECs and mural cells ( include pericytes and SMCs). To test our hypothesis, we propose the two Specific Aims:Aim 1. Define the role of exDll4 on brain vascular remodeling, cognative functions and related mechanisms in mice. Aim 2. Determine the role of monocyte derived Dll4 on brain vascular remodeling, cognitive funtion and mechanisms in LPS induced VCI mouse model using monocyte Dll4 (mDll4) KO mice. Accomplishing these aims will fill the knowledge gap regarding the mechanisms of the pathogenesis of VCI. 1