Joung-Hyun Lee

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P−) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P− equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

Atherosclerosis begins with the accumulation of cholesterol‐carrying lipoproteins on blood vessel walls and progresses to endothelial cell dysfunction, monocyte adhesion, and foam cell formation. Endothelialized tissue‐engineered blood vessels (TEBVs) have previously been fabricated to recapitulate artery functionalities, including vasoconstriction, vasodilation, and endothelium activation. Here, the initiation of atherosclerosis is emulated by designing branched TEBVs (brTEBVs) of various geometries treated with enzyme‐modified low‐density‐lipoprotein (eLDL) and TNF‐α to induce endothelial cell dysfunction and adhesion of perfused human monocytes. Locations of monocyte adhesion under pulsatile flow are identified, and the hemodynamics in the brTEBVs are characterized using particle image velocimetry (PIV) and computational fluid dynamics (CFD). Monocyte adhesion is greater at the side outlets than at the main outlets or inlets, and is greatest at larger side outlet branching angles (60° or 80° vs 45°). In PIV experiments, the branched side outlets are identified as atherosclerosis‐prone areas where fluorescent particles show a transient swirling motion following flow pulses; in CFD simulations, side outlets with larger branching angles show higher vorticity magnitude and greater flow disturbance than other areas. These results suggest that the branched TEBVs with eLDL/TNF‐α treatment provide a physiologically relevant model of early atherosclerosis for preclinical studies.

The immune system maintains a balance between protection and tolerance. Regulatory T cells (Tregs) function as a vital tolerance mechanism in the immune system to suppress effector immune cells. Additionally, Tregs can be utilized as a form of immunotherapy for autoimmune disorders. As T cells have previously been shown to exhibit sensitivity to the rigidity of an activating substrate upon activation via IL‐2 secretion, we herein explore the previously unknown effect of substrate rigidity on the induction of Tregs from conventional naïve mouse CD4⁺ T cells. Substrates with modulatable rigidities ranging from a hundred kilopascals to a few megapascals were fabricated via poly(dimethylsiloxane). We found that there was a significant increase in Treg induction at lower substrate rigidities (i.e., E ~ 100 kPa) compared to higher rigidity levels (i.e., E ~ 3 MPa). To confirm that this significant difference in induction rate was truly related to T‐cell mechanosensing, we administered compound Y‐27632 to inhibit myosin contractility. In the presence of Y‐27632, the myosin‐based contractility was disrupted and, as a result, the difference in Treg induction caused by the substrate rigidity was abrogated. This study demonstrates that mechanosensing is involved in Treg induction and raises questions about the underlying molecular mechanisms involved in this process. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3001–3008, 2018.

The immune synapse has emerged as a compelling example of structural complexity within cell-cell interfaces. This chapter focuses on the use of microcontact printing to isolate and investigate how spatial organization of signaling molecules drives the function of immune cells. In the process detailed here, multiple rounds of microcontact printing are combined to create patterned surfaces that control the relative spatial localization of CD3 and CD28 signaling in T cells, effectively replacing an antigen presenting cell with an engineered surface. A set of approaches used to address key issues of T cell activation are described and discussed.

Large-scale adaptive immune responses are often driven by minor populations of T cells. The system described here allows study of these rare cells by improving

Functional convergence of CD28 costimulation and TCR signaling is critical to T-cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, although the impact of this spatial organization on cell signaling remains unclear. We investigate this cross talk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4⁺ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T-cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4⁺ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment.

With conventional in vitro culture methods, it is difficult to study complex interactions of host cells with pathogens and drugs in physiologically relevant microenvironments. To simulate orthopaedic implant-associated infection, a multi-channel microfluidic device was used to (1) observe in real-time the development of osteoblasts into three-dimensional (3D) tissue-like structures and (2) study how this development was influenced by phenotypes of Staphylococcus epidermidis. In the absence of bacteria, osteoblasts formed a confluent layer on the bottom channel surface, gradually migrated to the side and top surfaces, and formed calcified 3D nodular structures in 8 days. The delivery timing and concentration of an antibiotic were controlled to produce small colony variants, sessile biofilms, or dead cells of S. epidermidis. In the presence of the small colony variants, osteoblasts initially adhered, and spread, but were killed within 2 days. In contrast, the sessile biofilms and dead bacteria cells did not significantly interfere with the formation of tissue-like structures. The results suggest the possibility of creating in vitro tissue-biofilm-biomaterial interfaces and therefore 3D tissue models, as an entirely new method of studying biofilm-related infection of orthopaedic implants with physiological relevance.