Abstract
Hyaluronic acid (HA), a nonsulfated glycosaminoglycan, regulates cell adhesion and migration. Small HA fragments (3-25 disaccharide units) induce neovascularization. We investigated the effect of HA and a HA fragment (10-15 disaccharide units, F1) on primary human endothelial cells (ECs). Human pulmonary ECs (HPAEC) and lung microvessel ECs (HMVEC-L) bound HA (K(d) approximately 1 and 2.3 nm, respectively) and expressed 17,780 and 16,690 HA binding sites, respectively. Both ECs showed HA-mediated cell adhesion; however, HMVEC-L was 1.5-fold better. Human umbilical vein ECs neither bound HA nor showed HA-mediated adhesion. All three ECs expressed CD44 ( approximately 110 kDa). The expression of receptor for HA-mediated motility (RHAMM) (approximately 80 kDa) was the highest in HMVEC-L, followed by HPAEC and human umbilical vein ECs. RHAMM, not CD44, bound HA in all three ECs. F1 was better than HA and stimulated a 2. 5- and 1.8-fold mitogenic response in HMVEC-L and HPAEC, respectively. Both HA and F1 induced tyrosine phosphorylation of p125(FAK), paxillin, and p42/44 ERK in HMVEC-L and HPAEC, which was blocked by an anti-RHAMM antibody. These results demonstrate that RHAMM is the functional HA receptor in primary human ECs. Heterogeneity exists among primary human ECs of different vascular origins, with respect to functional HA receptor expression and function.
Highlights
The integrity of vascular endothelium is crucial in several important processes, including maintenance of blood flow and blood vessel permeability and tone
To determine whether different vascular endothelial cells (ECs) differ in their ability to bind Hyaluronic acid (HA), we examined [3H]HA equilibrium binding in HMVEC-L, Human pulmonary ECs (HPAEC), and Human umbilical vein ECs (HUVEC)
Scatchard plot analyses indicate that both HPAEC and HMVEC-L contain a single class of high affinity HA binding sites with dissociation constants (Kd) of 1 and 2.3 nM, respectively
Summary
Primary Human EC Cultures—Human pulmonary artery ECs (HPAEC), human lung microvessel ECs (HMVEC-L), and HUVECs were obtained from Clonetics/BioWhittaker Inc., Walkersville, MD). To confirm that any differences observed in the intensity of bands, following immunoblot analysis, are not because of the differences in protein loading, prior to incubation with anti-RHAMM antibody, the blots were stained with Coomassie Blue and processed as described below. Ϳ80% confluent cultures of ECs grown on 6-well cultures plates were solubilized in RIPA buffer, and the cell lysates (prepared from 2 ϫ 105 cells) were immunoprecipitated by sequential incubations with anti-RHAMM antibody (1:200 dilution) at 4 °C for 16 h and goat anti-rabbit IgG-agarose beads at 4 °C for 90 min. The Coomassie Bluestained blots were photographed using a Kodak DC-20 camera that comes with gel analysis software (EDAS, Eastman Kodak Co.) Using this system, the intensity of protein bands in each sample lane with molecular masses between 83 kDa and 50 kDa and 50 kDa and 36 kDa were compared for detecting differences in protein loading and possible artifacts during protein transfer. Both strands of the cloned cDNA insert were sequenced in the University of Miami’s DNA core facility, using an automated DNA sequencer and T7 and SP6 universal primers
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