Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro

Abstract

The development of vessel-like structures in vitro to mimic as well as to realize the possibility of tissue-engineered small vascular networks presents a major challenge to cell biologists and biotechnologists. We aimed to establish a three-dimensional (3-D) culture system with an endothelial network that does not require artificial substrates or ECM compounds. By using human skin fibroblasts and endothelial cells (ECs) from the human umbilical vein (HUVECs) in diverse spheroid coculture strategies, we verified that fibroblast support and modulate EC migration, viability, and network formation in a 3-D tissue-like stromal environment. In mixed spheroid cultures consisting of human ECs and fibroblasts, a complex 3-D network with EC tubular structures, lumen formation, pinocytotic activity, and tight junction complexes has been identified on the basis of immunohistochemical and transmission electron microscopic imaging. Tubular networks with extensions up to 400 m were achieved. When EC suspensions were used, EC migration and network formation were critically affected by the status of the fibroblast. However, the absence of EC migration into the center of 14-day, but not 3-day, precultured fibroblast spheroids could not be attributed to loss of F viability. In parallel, it was also confirmed that migrated ECs that entered cluster-like formations became apoptotic, whereas the majority of those forming vessel-like structures remained viable for Ͼ8 days. Our protocols allow us to study the nature of tubule formation in a manner more closely related to the in vivo situation as well as to understand the basis for the integration of capillary networks in tissue grafts and develop methods of quantifying the amount of angiogenesis in spheroids using fibroblast and other cells isolated from the same patient, along with ECs. endothelium; angiogenesis; human umbilical vein endothelial cell; multicellular spheroid; coculture; tubular structures ANGIOGENESIS IS A COMPLEX morphogenetic process initiated primarily by sprouting of endothelial capillaries from existing blood vessels to form an endothelial plexus. After being remodeled, the vasculature matures by recruitment of perivascular cells and smooth muscle cells. Vascular maturation attenuates the rate of vascular sprouting and prevents vascular collapse and regression (18,24), which is partly reflected by contact inhibition of endothelial cell (EC) proliferation in coculture systems with pericytes or smooth muscle cells (7,12,29,47). In adults, the angiogenic process is essential during wound healing, tissue repair, and remodeling and for female reproductive cycles, among many normal and pathological conditions. Mechanistic studies designed to gain a better understanding of, and an ability to manipulate, the angiogenic process in health and disease have intensified during the past 30 years, having been stimulated by Folkman's (2,16,19,25,27) initial hypotheses regarding the angiogenic switch during tumor cell growth and the development of the concept of antiangiogenic ther...

A novel physiological culture system that mimics luteal angiogenesis

“…Furthermore, the development of vasculature in vitro was stimulated by physiological doses of LH, FGF2 and VEGFA. While a number of cell culture systems have been developed to investigate angiogenesis, the vast majority use a combination of cell lines such as human umbilical vein endothelial cells (HUVECs) or bovine aortic endothelial cells (BAEC) with human fibroblasts (Bishop et al 1999, Kunz-Schughart et al 2006) rather than primaryderived cells. While these systems provide an invaluable tool to screen potential pro-and anti-angiogenic compounds for use in tissue repair and treatment of diseases such as cancer, they are less relevant to study the physiological mechanisms controlling angiogenesis.…”

Fibroblast-Endothelial Partners for Vascularization Strategies in Tissue Engineering

“…24 Understanding this process remains a challenge, since fibroblasts have the capacity to alter the mechanical extracellular microenvironment, thereby regulating vascularization processes. 25 Fibroblast-derived proteins, including growth factors and matrix proteins, have been shown to modulate EC sprouting and the expansion of capillary-like networks in vitro, [26][27][28] contributing to the role of fibroblasts as periendothelial cells in vivo. 29 Thus, the hypothesis underlying herein is that, when cocultured with OECs or mature ECs, different types of fibroblasts will exert distinct influences in the assembly of capillary-like structures.…”

Transcriptional Profiling Reveals Crosstalk Between Mesenchymal Stem Cells and Endothelial Cells Promoting Prevascularization by Reciprocal Mechanisms

“…Tissue engineering-based research in both two-dimensional (2D) and three-dimensional cocultures in vitro revealed that the active interaction between ECs and MSCs (or osteoprogenitor and fibroblast) induced the formation of tube-like cell aggregation structures. This result indicates that angiogenesis and prevascularization are involved in the cellcell communication and remodeling of the functional blood vessel development in vivo [5,[18][19][20][21][22][23][24][25][26]. Moreover, MSCs could repress ECs by regulating cytokine-induced leukocyte recruitment [27], while the activated ECs could regulate the MSCs-to-ECs transmigration in a leukocyte-like mechanism [28,29].…”