Ruliffson Brian N K, Larson Stephen M, Xhupi Eleni K, Herrera-Diaz Diana L, Whittington Catherine F
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA 01609, USA.
Department of Biomedical Engineering and Chemical Engineering, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Lymphatics. 2024 Sep;2(3):177-194. doi: 10.3390/lymphatics2030015. Epub 2024 Sep 19.
Despite chronic fibrosis occurring in many pathological conditions, few in vitro studies examine how fibrosis impacts lymphatic endothelial cell (LEC) behavior. This study examined stiffening profiles of PhotoCol-commercially available methacrylated type I collagen-photo-crosslinked with the photoinitiators: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959 (IRG), and Ruthenium/Sodium Persulfate (Ru/SPS) prior to evaluating PhotoCol permeability and LEC response to PhotoCol at stiffnesses representing normal and fibrotic tissues. Ru/SPS produced the highest stiffness (6 kilopascal (kPa)) for photo-crosslinked PhotoCol, but stiffness did not change with burst light exposures (30 and 90 s). The collagen fibril area fraction increased, and dextran permeability (40 kilodalton (kDa)) decreased with photo-crosslinking, showing the impact of photo-crosslinking on microstructure and molecular transport. Human dermal LECs on softer, uncrosslinked PhotoCol (0.5 kPa) appeared smaller with less prominent vascular endothelial (VE)-cadherin (cell-cell junction) expression compared to LECs on stiffer PhotoCol (~6 kPa), which had increased cell size, border irregularity, and VE-cadherin thickness (junction zippering) that is consistent with LEC morphology in fibrotic tissues. Our quantitative morphological analysis demonstrates our ability to produce LECs with a fibrotic phenotype, and the overall study shows that PhotoCol with Ru/SPS provides the necessary physical properties to systematically study LEC responses related to capillary growth and function under fibrotic conditions.
尽管慢性纤维化发生于多种病理状况中，但很少有体外研究探讨纤维化如何影响淋巴管内皮细胞（LEC）的行为。本研究在评估PhotoCol在代表正常组织和纤维化组织的硬度下的通透性以及LEC对PhotoCol的反应之前，检测了用苯基-2,4,6-三甲基苯甲酰基膦酸锂（LAP）、Irgacure 2959（IRG）和钌/过硫酸钠（Ru/SPS）等光引发剂进行光交联的市售甲基丙烯酸化I型胶原（PhotoCol）的硬化情况。Ru/SPS使光交联的PhotoCol产生了最高的硬度（约6千帕斯卡（kPa）），但硬度并未随突发光照（30秒和90秒）而改变。光交联使胶原纤维面积分数增加，葡聚糖通透性（40千道尔顿（kDa））降低，显示了光交联对微观结构和分子运输的影响。与在较硬的PhotoCol（约6 kPa）上的LEC相比，在较软的未交联PhotoCol（约0.5 kPa）上的人真皮LEC显得更小，血管内皮（VE）-钙黏蛋白（细胞间连接）表达不那么突出，而在较硬的PhotoCol上的LEC细胞大小增加、边界不规则且VE-钙黏蛋白厚度增加（连接拉链化），这与纤维化组织中的LEC形态一致。我们的定量形态学分析证明了我们产生具有纤维化表型的LEC的能力，并且整体研究表明，用Ru/SPS处理的PhotoCol提供了必要的物理特性，以系统地研究纤维化条件下与毛细血管生长和功能相关的LEC反应。
