Premium
Interstitial Collagen IV: A template for pericyte recruitment
Author(s) -
Hoque Maruf,
Chappell John
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02976
Subject(s) - extracellular matrix , microbiology and biotechnology , chemistry , basement membrane , small hairpin rna , transfection , pericyte , vascular smooth muscle , protein subunit , medicine , biology , endothelial stem cell , gene , biochemistry , in vitro , gene knockdown , smooth muscle
During an acute ischemic stroke, the integrity of the cerebrovascular wall is compromised (Hu et al. 2017). This specialized vessel barrier is formed through coordination between endothelial cells (ECs) and pericytes (PCs) and involves extracellular matrix (ECM) proteins comprising the vascular basement membrane (vBM). A component of the vBM, Type IV Collagen (Col‐IV), modulates cell adhesion and migration with the Col4a1 subunit in particular regulating angiogenic remodeling. Aberrant Col4a1 expression in humans has been linked to diseases such as stroke (Alamowitch et al. 2009). Herein, we utilize a murine embryonic stem cell (ESC) model to investigate how perturbations in Col‐IV deposition, and specifically with respect to Col4a1, impacts PC‐EC interactions during vascular development. Col‐IV was targeted by (i) administering α,α‐dipyridyl (an iron chelator that disrupts collagen fibril formation, Ikeda et al. 1992) or (ii) plasmid transfection (pLKO.1) with shRNA insert targeting Col4a1 (pLKO‐Col4a1; MISSION shRNA Plasmid DNA, Sigma). This study aims to inform rational drug design for promoting cerebrovascular stability post‐acute ischemic stroke. In the current study, we administered α,α‐dipyridyl on days 6 and 8 of ESC differentiation into primitive vascular structures and cultured these vessel networks for 10 days. We quantified confocal images of vessels and Col‐IV content, finding a ~20% increase in Col‐IV density, with a 5% increase in EC networks with the 25µM dose. Application of a 50µM dose, however, yielded a ~60% decrease in Col‐IV and EC densities, when compared to time‐matched, untreated controls. The 25µM dose did not appear to affect ESC‐derived vessel formation but altered PC recruitment to the emerging endothelium. Additionally, we observed that a subset of cells in these cultures appeared to reactively overproduce Col‐IV with the 25µM dose, presumably to prevent vascular degradation. The majority of Col‐IV signal appeared to be associated with EC in all scenarios, with a reduction in the PC reporter signal (Ng2:DsRed) when α,α‐dipyridyl was administered, suggesting that interstitial Col‐IV (i.e. Col‐IV beyond the vessel wall) may contribute to successful PC recruitment. In separate experiments, we transfected ESCs with a pLKO.1‐Col4a1 plasmid 7 days after differentiation and cultured ESC‐derived vessels for 8 days. Quantification of confocal images revealed a 19.3% reduction in Col‐IV content and a 93.6% reduction in Col4a1 via qRT‐PCR. The Col‐IV reduction do not appear to be spatially localized to ECs, indicating that interstitial Col‐IV is more likely to be reduced first following transfection. Furthermore, we observed a reduction in the PC reporter signal upon the pLKO.1‐Col4a1 transfection, further suggesting that interstitial Col‐IV may be involved in PC recruitment to vessel ECs. Next steps include testing lower α,α‐dipyridyl doses (5, 10, 15, & 20µM) and Col4a1 targets (using different pLKO.1‐Col4a1 plasmids) to establish conditions in which interstitial Col‐IV is reduced at different levels and EC‐associated Col‐IV can also be reduced. We will also pursue similar experiments with a focus on Col3a1 as our lab and others have found that Col3a1 may be relevant to vascular stability (Frank et al. 2015) and PC recruitment mechanisms.