A composite model of the human postcapillary venule for investigation of microvascular leukocyte recruitment

Abstract

Neutrophil extravasation occurs across postcapillary venules, structures composed of endothelial cells (ECs), pericytes (PCs), and basement membrane (BM). We constructed composite models of the human postcapillary venule, combining ECs with PCs or PC-deposited BM, to better study this process. Quiescent and tumor necrosis factor α (TNF-α)-activated composites demonstrated in situ-like expression of cadherins, E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet-endothelial cell adhesion molecule 1 (PECAM-1), CD99, and interleukin 8 (IL-8). After TNF-α activation, the ECs supported greater neutrophil adhesion (66.1 vs. 23.7% of input cells) and transmigration (35.1 vs. 7.20% of input cells) than did the PCs, but the composites behaved comparably (no significant difference) to ECs in both assays. TNF-α-activated EC-conditioned medium (CM) increased transmigration across the PCs, whereas TNF-α-activated PC-CM decreased transmigration across the ECs, and culturing on PC-derived BM decreased both adhesion to and transmigration across the ECs. Anti-very late antigen 4 (VLA-4; on neutrophils) inhibited adhesion to TNF-α-activated composites, but not to ECs alone. Anti-CD99 (expressed on all 3 cell types) inhibited transmigration across the composites (14.5% of control) more than across the ECs (39.0% of control), and venular shear stress reduced transmigration across the ECs (17.3% of static) more than across the composites (36.7% of static). These results provide proof of concept that our composite human EC/PC/BM venular construct can reveal new interactions in the inflammatory cascade.

Proteomic Analysis of the Pericyte Derived Extracellular Matrix

“…1,20,24 Briefly, a human placenta was segmented, digested with collagenase, and the resulting cells were cultured in M199 (Gibco, Grand Island, NY, USA) medium supplemented with 20% FBS (Hyclone Laboratories, Logan, UT, USA), and 1% penicillin-streptomycin (Gibco). Isolated pericytes are routinely screened by flow cytometry to confirm expression of NG2, CD90, CD146, PDGFR-b and a-SMA.…”

“…Additionally, isolated PC lack SMC markers (SMMHC), endothelial cell markers (CD31, CD34), or leukocyte markers (CD45) as previously described. 1,20,24,34 Detection of these markers was performed by flow cytometry analysis, prior to which the cells were trypsinized, immediately fixed in 4% paraformaldehyde (PFA), and blocked with 2% bovine serum albumin (BSA). The samples were stained against NG2 (Clone H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD90 (Clone K-15, Santa Cruz), CD146 (Clone P1H12, Biolegend, San Diego, CA, USA), PDGFR-b (Clone 18A2, Santa Cruz), a-SMA (Clone 1A4, Santa Cruz), SMMHC (Clone N-16, Santa Cruz), CD31 (Clone C20, Santa Cruz), CD34 (Clone 581, Sigma, St. Louis, MO, USA) and CD45 (Clone MEM-28, Sigma).…”

“…The cells were cultured on flasks coated with 0.1% gelatin and supplemented with M199 medium containing 20% FBS, 1% penicillin-streptomycin, and 50 mg/mL endothelial cell growth supplement (Collaborative Biomedical Products, Bedford, MA, USA) as described. 1,20,34 …”

“…Further, placental isolated PC do not express smooth muscle myosin heavy chain (SMMHC), an SMC marker, CD31 and CD34, EC markers, or CD45, a leukocyte marker, as previously described. 1,20,24,34 The isolated PC populations demonstrate a distinct genotype when compared to mesenchymal stem cells since they lack the expression of 21 out of 65 strongly expressed mesenchymal stem cell markers. 24 By characterizing the composition of PC derived ECM, we provide a foundation supporting the use of PC ECM as an alternate scaffold for tissue engineered constructs that require vascularization and as an experimental matrix for studies of vascular basement membrane function.…”

Pericytes in Chronic Lung Disease

“…Subsequent studies from the same research group improved on this model by incorporating a pericyte-deposited basement membrane, with ECs and pericytes seeded on opposing sides of a Transwell membrane. Interestingly, in the presence of a basement membrane, EC/pericyte composites performed comparably to ECs alone following activation with TNF-α [40]. Other studies have shown that pericytes can further contribute to coordinating the immune response by responding to damage-associated molecular patterns such as necrotic cell lysates or TNF-α.…”