Inorganic phosphate (Pi) is required for cellular function. It is required for kinase signaling, DNA and lipid formation and function, as well as ATP, which is an essential component of energy metabolism. Excess extracellular Pi can have several negative effects, including accelerated ectopic calcification, increased oxidative stress, and abnormal signal transduction [1]. This response to changes in extracellular Pi levels suggests that mammalian cells can detect and adopt to Pi availability. At the organ level, the kidneys are primarily responsible for Pi metabolism. Pi homeostasis is maintained by increasing urinary Pi excretion in response to dietary or blood Pi changes. This balance is impacted by multiple organ systems, including the kidneys, intestines, bones, and parathyroid glands [[2], [3], [4]].

Pi homeostasis in the brain is disrupted in primary brain calcification (PBC). SLC20A2 mutations are known to cause PBC [5]. In the brain, Pi is transported from the extracellular to the intracellular space primarily by the type III sodium-dependent Pi transporters, SLC20A1 and SLC20A2. In both mice and humans, we discovered that SLC20A2 is expressed in neurons, astrocytes, and endothelial cells in the central nervous system [6]. PBC pathogenesis is caused by the calcification of cerebral small vessels, composed of endothelial cells and vascular smooth muscle cells [7]. Pathogenic mutations of PBC in these vascular cells include platelet-derived growth factor subunit B (PDGFB) and junctional adhesion molecule 2 (JAM2) [8,9]. PDGFB stabilizes vascular structure by interacting with PDGFRβ expressed on pericytes. Biallelic mutation of JAM2 is associated with brain calcification due to the clinical study of unrelated four families, suggesting that defects in intercellular adhesion are an important mechanism for brain calcification [9].

Among the cells that form cerebral blood vessels, vascular smooth muscle cells have been extensively studied in the field of vascular calcification. In cultured senescent vascular smooth muscle cells, osteoblast marker genes were overexpressed, and the knockdown of these genes reduced calcification. These findings indicate that vascular smooth muscle cell dysfunction is a significant contributor to calcification [10,11]. Microvascular calcification of SLC20A2 knockout mice has also been observed in pericytes, which have functional similarity to vascular smooth muscle cells, indicating that calcifying cells are not endothelial cells [12,13]. The effect of disruption of Pi homeostasis on endothelial cell function is unknown. Endothelial cells play an important role in keeping the BBB functional and regulating cerebral blood flow [14]. BBB disintegration has been linked to the pathogenesis of neurodegenerative diseases such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis [15].

The current study also aims to investigate the effect of the genetically knockdown of SLC20A1 and SLC20A2 on the phenotypes of human brain microvascular endothelial cells (hBMECs). Studying the phenotypic changes in SLC20A2-silenced hBMECs will provide evidence for the Pi's role in cerebrovascular pathology, not only in PBC but also in other neurodegenerative diseases.