SPARC (secreted protein acidic and rich in cysteine) regulates endothelial cell shape and barrier function.

Abstract

SPARC (secreted protein acidic and rich in cysteine) can be selectively expressed by the endothelium in response to certain types of iqjury and induces rounding in adherent endothelial cells in vitro. To determine whether SPARC might influence endothelial permeability, we studied the effect of exogenous SPARC on the movement of 14C-labeled bovine serum albumin across postconfluent bovine pulmonary artery endothelial cells. SPARC increased (P < 0.02) transendothelial albumin flux in a dose-dependent manner at concentrations -0.5 jug/ml. At a fixed dose (15 pg/mi), exposure times > 1 h augmented (P < 0.005) albumin flux by 1.3-to 3.6-fold; this increase was blocked by anti-SPARC antibodies but not by inhibition of protein synthesis. Barrier dysfunction was not associated with loss of cell viability. Monolayers exposed to SPARC exhibited a rounded morphology and intercellular gaps. Prior stabilization ofF-actin with phallicidin protected against the changes in barrier function (P = 0.0001) that were otherwise induced by SPARC. Bovine aortic and retinal microvascular endothelia also responded to SPARC. We propose that SPARC regulates endothelial barrier function through F-actin-dependent changes in cell shape, coincident with the appearance of intercellular gaps, that provide a paracellular pathway for extravasation of macromolecules.The vascular endothelium presents a selective barrier that actively regulates movement of circulating macromolecules and cells into extravascular tissues and compartments (1, 2). Although mechanisms regulating this endothelial barrier are not well understood, a structure-function relationship appears to exist between endothelial cell (EC) shape and barrier function. Specific agonists can induce changes in EC shape coincident with the formation of intercellular gaps, which in turn provide a paracellular pathway for the flux of macromolecules (3-5). Actin organization is postulated to regulate EC shape as well as barrier function (5)(6)(7)(8)(9)(10)(11)(12). Actin filamentdisrupting agents increase endothelial permeability (6), prior stabilization of actin protects against increases in permeability (7-9), and mediators of permeability have been shown to induce cytoskeletal rearrangement (8-12 (20).Assay of Albumin Flux. EC were grown to confluence in 0.5 ml of medium on gelatin-impregnated polycarbonate filters (13-mm diameter, 0.4-,im pore size; Nucleopore) mounted in chemotactic chambers (ADAPS, Dedham, MA) as described (4). These chambers, which served as the upper compartment for the assay chambers, were inserted into wells of 24-well plates, each well containing 1.5 ml of medium and serving as the lower compartment of the assay chamber. 14C-BSA (Sigma; 30.1 pCi/mg of protein; 1 uCi = 37 kBq), the tracer molecule, was prepared in serum-free medium supplemented with BSA (34 mg/ml) to produce a final protein concentration equivalent to medium enriched with 10%o FBS. The baseline barrier function of each monolayer was established by application of an equivalent amount of 14C-BS...

Tumour-derived SPARC drives vascular permeability and extravasation through endothelial VCAM1 signalling to promote metastasis

“…1). Early studies have shown a role for SPARC in vascular biology in non-malignant settings 21,41 . However, its importance in the regulation of tumour-initiated vascular endothelial permeability has remained poorly studied.…”

“…Other proteins differentially expressed in 1205Lu CM versus 501Mel included factors involved in additional important tumour cell functionalities such as angiogenesis (NRP1), fibrinolysis (PAI1, GDN, UPAR) or associated with extracellular microvesicles and exosomes (HS90B, RAB7A). The discovery that SPARC, a matricellular protein involved in tumour cell invasion and survival 20 , and endothelial barrier function 21 , was 10-fold more abundant in metastatic melanoma cell CM as compared with CM from a poorly metastatic cell prompted us to investigate whether SPARC contributes to paracrine induction of vascular permeability by melanoma cellderived CM. We, therefore, engaged different approaches to manipulate SPARC expression and function.…”

Novel markers for poor prognosis in head and neck cancer

“…17 This causes degradation of both interstitial and basement membrane matrices 18 and increases endothelial permeability with the appearance of intercellular gaps, which provide a pathway for extravasation of tumor cells. 19 The exact role of SPARC in tumor angiogenesis is still unclear but may include a role in VEGF (vascular endothelial growth factor) functions and in extravasation of tumors cells mediated by increased permeability of the endothelial barrier. 17 SPARC overexpression has previously been associated with human tumors.…”

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

“…This emphasis on singlecell morphology and on the cell-matrix interface has encouraged studies of cells under subconfluent conditions. Only recently has it been appreciated that counteradhesive proteins may also influence EC-EC interactions (Goldblum et al, 1994a).…”