Elsevier

Microvascular Research

Volume 68, Issue 3, November 2004, Pages 258-264
Microvascular Research

Interstitial flow differentially stimulates blood and lymphatic endothelial cell morphogenesis in vitro

https://doi.org/10.1016/j.mvr.2004.08.002Get rights and content

Abstract

Whereas high shearing flows are known to induce endothelial cell remodeling, we show here that very low interstitial flow rates trigger endothelial cell morphogenesis in 3D cultures. Interstitial flow is a functionally critical component of the circulation, and we have recently observed that it plays a regulatory role in lymphangiogenesis; here we investigate interstitial flow as a powerful morphoregulatory stimulant. We exposed both lymphatic and blood endothelial cells (LECs and BECs) to interstitial flow in 3D collagen gels as well as simple shear flow in 2D monolayers. We found that under interstitial flow (average 10 μm/s for 6 days), both cell types underwent drastic morphologic changes from static conditions: LECs formed large vacuoles and long extensions, while BECs formed multicellular branched lumen-containing networks. Under planar shear (20 dyn/cm2 for 24 h), LECs downregulated their cell–cell adhesions compared to BECs but did not differ morphologically; both aligned with flow as expected. The significance of these findings is twofold: first, they identify an important role of interstitial flow for in vitro microvascular organization and stabilization, and second, they demonstrate for the first time notable differences between LEC and BEC response to the biophysical environment, reflecting some of their functional differences in vivo.

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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