Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro
Introduction
The circulation is a convective transport system, and as such, the blood and lymphatic microvasculature are constantly subjected to fluid stresses. The two tissue types serve complementary functions, but they are structurally and developmentally distinct. Blood capillary endothelium experiences fluid shear stress on its luminal side with little transvascular flow due to tight cell–cell junctions and relatively low vessel permeability. In contrast, the lymphatic capillary endothelium is exposed to interstitial fluid stresses as it drains interstitial fluid with loose cell–cell junctions and very high permeability; these properties facilitate interstitial protein convection (Schmid-Schönbein, 1990).
Interstitial flow is present to some degree in all tissues and, importantly, constitutes the biophysical environment in developing vascular networks and intussusceptive processes as well as in tissues undergoing lymphatic capillary development. We have recently shown that interstitial flow is important for lymphangiogenesis in a mouse model of regenerating skin (Boardman and Swartz, 2003), but to date, the specific role of interstitial flow on either blood or lymphatic capillary morphogenesis has not been explored in vitro. However, it is well accepted that mechanical forces such as fluid shear (McCormick et al., 2003, Shyy and Chien, 2002) and matrix strain (Ingber and Folkman, 1989, Korff and Augustin, 1999) take part in regulating blood capillary morphogenesis and endothelial cell morphology.
To elucidate and compare the morphological and organizational responses of microvascular lymphatic and blood endothelial cells (LECs and BECs) to interstitial fluid flow, we utilized a recently developed interstitial flow system for 3D collagen gel cultures (Ng and Swartz, 2003). We show here that interstitial flow is a morphogenetic mediator of microvascular organization that is distinctly different from that of monolayer shear stress, which is well known to align endothelial cells and activate several genetic events (Dewey et al., 1981, Franke et al., 1984, McCormick et al., 2003). Our findings also identify key differences between LECs and BECs—not in the genes they express, as has been recently published (Makinen et al., 2001, Podgrabinska et al., 2002)—but instead in their functional cell–cell and cell–matrix interactions and in the different ways they respond to biophysical environmental stimuli, indicating different biophysically mediated mechanisms of morphogenesis in development and remodeling.
Section snippets
Culture of human microvascular LECs and BECs
Primary cultures of LECs and BECs (passage 7–9) isolated from human neonatal foreskins using a LYVE-1 antibody (Podgrabinska et al., 2002) were a kind gift of Dr. Mihaela Skobe (Mt. Sinai School of Medicine, NY). Cells were cultured on collagen-coated dishes in EC basal medium (Cambrex BioScience, Walkersville, MD) supplemented with 20% FBS (Gibco–InVitrogen, Carlsbad, CA), 1% penicillin–streptomycin–amphotericin B, 50 μM DBcAMP, and 1 μg/ml hydrocortisone acetate (all from Sigma, St. Louis,
LECs vs. BECs in static 3D cultures
In static 3D collagen cultures, LECs maintained better survival than BECs and tolerated lower (2%) serum concentrations (Fig. 1). This is consistent with in vivo conditions where BECs are exposed to blood serum directly while LECs are bathed in plasma filtrate. PMA, a protein kinase C activator that is also known to induce microvascular endothelial cell tubulogenesis in vitro (Montesano and Orci, 1985), caused both the LECs and BECs to similarly rearrange into networks of capillary-like
Acknowledgments
The authors are grateful to William Russin for assistance with reflectance microscopy, Mihaela Skobe and Simona Podgrabinska for providing LECs and BECs, and the Whitaker and National Science Foundations for financial support.
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