The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cellderived human brain microvascular endothelium interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.

Abstract-Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H 2 O 2 and O 2 Ϫ production in ECs. Next, we used ECs obtained from p47phox Ϫ/Ϫ mice (MAE-p47 Ϫ/Ϫ ), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47 Ϫ/Ϫ , but it was restored when the cells were transfected with p47 phox plasmid. Moreover, OS-induced O 2 Ϫ production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. Key words: BMP4 Ⅲ oscillatory shear Ⅲ reactive oxygen species Ⅲ monocyte adhesion Ⅲ endothelial cells Ⅲ NADPH oxidase V ascular endothelial cells (ECs) are constantly exposed to fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, the regions of branched and curved arteries exposed to disturbed flow conditions including oscillatory shear stress (OS) correspond to "lesion-prone areas" that preferentially develop atherosclerosis. 1,2 In contrast, straight arteries exposed to steady, high levels of laminar shear stress (LS) are relatively well protected from atherosclerotic plaque development. 1,2 Atherosclerosis is an inflammatory disease preferentially occurring in lesion-prone areas. 2,3 The earliest measurable markers of atherogenesis include expression of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and subsequent monocyte adhesion and recruitment into the lesion-prone areas. 2,4,5 Additional critical atherogen...

The brain vasculature maintains brain homeostasis by tightly regulating ionic, molecular, and cellular transport between the blood and the brain parenchyma. These blood-brain barrier (BBB) properties are impediments to brain drug delivery, and brain vascular dysfunction accompanies many neurological disorders. The molecular constituents of brain microvascular endothelial cells (BMECs) and pericytes, which share a basement membrane and comprise the microvessel structure, remain incompletely characterized, particularly in humans. To improve the molecular database of these cell types, we performed RNA sequencing on brain microvessel preparations isolated from snap-frozen human and mouse tissues by laser capture microdissection (LCM). The resulting transcriptome datasets from LCM microvessels were enriched in known brain endothelial and pericyte markers, and global comparison identified previously unknown microvessel-enriched genes. We used these datasets to identify mouse-human species differences in microvessel-associated gene expression that may have relevance to BBB regulation and drug delivery. Further, by comparison of human LCM microvessel data with existing human BMEC transcriptomic datasets, we identified novel putative markers of human brain pericytes. Together, these data improve the molecular definition of BMECs and brain pericytes, and are a resource for rational development of new brain-penetrant therapeutics and for advancing understanding of brain vascular function and dysfunction. The blood-brain barrier (BBB) regulates blood flow, supplies the brain with nutrients, and facilitates clearance of a variety of substances. The BBB is comprised of brain microvascular endothelial cells (BMECs), the principal barrier-forming cell. BMECs are also intimately associated with brain pericytes, mural cells that line the outside of microvessels and are linked to endothelial cells by a shared vascular basement membrane 1. The BBB is required to maintain brain homeostasis, but also prevents clinically relevant doses of many therapeutics from entering the brain 2,3. Brain vascular dysfunction plays a role in several neurological disorders, including some with cellautonomous defects in BMEC or pericyte function 4-7. Due to its role in neurological disorders and important implications for brain drug delivery, the brain vasculature has been the subject of intense research, often focused on identifying mechanisms underlying its unique behavior. Our understanding of brain vascular development, function, dysfunction, and molecular constituents, however, has been advanced largely by mouse models. The scarcity of human brain tissue and low abundance of brain vascular cells has limited molecular profiling of the human brain vasculature. Improved molecular understanding of human BMECs and pericytes could aid in the development of new BBB-penetrant therapeutics and advance new hypotheses about mechanisms of brain vascular dysfunction in disease. Mouse brain vascular cells have previously been isolated and transcriptionally prof...