Cells were incubated in the presence of mouse anti-Cluster of Differentiation (CD) 31 antibody (1:200; cat

Cells were incubated in the presence of mouse anti-Cluster of Differentiation (CD) 31 antibody (1:200; cat. cathepsin-induced endothelial cell invasion, partially via suppressing the expression and activity of cathepsin L and S. The findings of the present study suggested that the potent anti-angiogenic properties of palmitate may be mediated by cysteine proteases. (3) demonstrated that cysteine protease cathepsin L-deficient mice presented impaired wound recovery, indicating an important role for cathepsin L in neovascularization. Furthermore, the study validated the ability of cathepsin L to induce angiogenesis, as transferred cathepsin L-deficient progenitor cells did not migrate to ischemic areas or augment vasculogenesis. Conversely, forced expression of cathepsin L in mature endothelial cells markedly enhanced cell invasion. Furthermore, Shi (4) demonstrated that cathepsin S was required for microvessel formation. Cysteine proteases have previously been demonstrated to be important in apoptosis and cell survival, KRIBB11 separate from their role in proteolysis of extracellular matrix in vascular remodeling (5C7); however, the underlying mechanism of this process remains to be fully elucidated. Angiogenesis is associated with atherosclerosis, and numerous risk factors of atherosclerosis, including diabetes and insulin resistance, are accompanied by high levels of free fatty acids (FFA) (8,9). However, the specific role of FFAs and their association with angiogenesis remains to be elucidated. FFAs have been demonstrated to exert effects on endothelial cells via enhancing reactive oxygen species levels or impairing nitric oxide production (10); therefore, increased levels of FFA will inhibit angiogenesis. Palmitate, which is the most frequently occurring form of saturated FFA present in human serum, contributes to lipotoxicity (11). In addition to the aforementioned characteristics, palmitate has been detected to induce apoptosis KRIBB11 in a variety of tissues (10,12,13) and decrease cardiolipid synthesis, resulting in the release of cytochrome c (14). Cathepsin L and S have therefore been confirmed to be important in endothelial cell angiogenesis; however, it remains to be elucidated as to whether FFA levels may influence cathepsin-mediated angiogenesis. The present study examined the proliferation, apoptosis and invasion of human umbilical vein endothelial cells (HUVECs) following exposure to palmitate in the presence or absence of selective cathepsin inhibitors, and observed that palmitate impaired cathepsin protein expression levels and activity. Materials and methods Cell culture and incubation with fatty acids HUVECs were purchased from the American Type Culture Collection (Manassas, VA, USA; PCS-100-010) and cultured in M199 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 20% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences) at 37C in an atmosphere containing under 5% CO2. Cells were passaged every 2C3 days once they reached maximum confluence. Cells were incubated in M199/10% FBS medium supplemented with 0.05, 0.1, 0.2, 0.4 or 0.6 mM palmitate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) preconjugated with FFA-free bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at a 1:1 molar ratio. Control cells were grown with the same medium containing FFA-free BSA. If not stated otherwise, cells were incubated for 1 h with 10 cathepsin L inhibitor (z-FF-FMK; cat. no. 219421; Calbiochem; EMD Millipore, Billerica, MA, USA) and cathepsin S inhibitor (z-FL-COCHO.H2O; cat. no. 219393; Calbiochem; EMD Millipore) at 37C, which was followed IL8 by incubation with palmitate or FFA-free BSA for 24 h at 37C. Immunofluorescence staining HUVECs were fixed in 4% paraformaldehyde for 20 min and incubated at 37C in blocking buffer (PBS containing 5% BSA). Cells were incubated in the presence of mouse anti-Cluster of Differentiation (CD) 31 antibody (1:200; cat. no. SC-81158; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at 37C and washed three times in PBS. Cells were subsequently incubated with rhodamine-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:1,000; cat. no. 31660; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 1 h at 37C. Nuclei were stained with DAPI (1:10,000; Invitrogen; Thermo Fisher Scientific, Inc.) and were examined with an Olympus IX70 inverted fluorescence microscope. Cell proliferation assay The effect of elevated palmitate concentration on HUVEC proliferation was analyzed utilizing Cell Counting kit (CCK)-8 KRIBB11 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer’s protocol. The amount of the formazan dye generated in cells was directly proportional to the number of living cells. Absorbance of the samples was measured at a wavelength of 450 nm. Cell apoptosis assay HUVECs were grown in M199 medium and pretreated for 1 h at 37C with indicated protease inhibitors prior to the addition of 0, 0.05, 0.1, 0.2, 0.4 or 0.6.