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 37°C in an atmosphere containing under 5% CO₂. Cells were passaged every 2–3 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 µΜ 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 37°C, which was followed by incubation with palmitate or FFA-free BSA for 24 h at 37°C.

HUVECs were fixed in 4% paraformaldehyde for 20 min and incubated at 37°C 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 37°C 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 37°C. Nuclei were stained with DAPI (1:10,000; Invitrogen; Thermo Fisher Scientific, Inc.) and were examined with an Olympus IX70 inverted fluorescence microscope.

The effect of elevated palmitate concentration on HUVEC proliferation was analyzed utilizing Cell Counting kit (CCK)-8 (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.

HUVECs were grown in M199 medium and pretreated for 1 h at 37°C with indicated protease inhibitors prior to the addition of 0, 0.05, 0.1, 0.2, 0.4 or 0.6 mM palmitate. Following palmitate treatment for 24 h at 37°C, cells were harvested. A total of 1×10⁶ cells were suspended in 200 µl Annexin V binding buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated with 2.5 µl annexin V-fluorescein isothiocyanate (FITC) and 2.5 µl propidium iodide (PI; BD Biosciences) for 15 min at room temperature in the dark. Unstained cells and those stained with Annexin V-FITC or PI alone were used as controls to set up compensation and quadrants. Cell apoptosis was investigated by flow cytometry within 30 min after fluorescent labeling by using FlowJo^® software (version 8.8.7; Tree Star, Inc., Ashland, OR, USA). Results were determined as the percentage of cells that were early (Annexin V⁺PI⁻) or late (Annexin V⁺PI⁺) apoptotic.

HUVEC invasion across a polycarbonate membrane containing 8-µm pores was performed with Transwell 24-well plates according to the manufacturer's protocol. Briefly, the membrane was precoated with type I collagen (1 mg/ml; Sigma-Aldrich; Merck KGaA). Detached cells (1×10⁵ cells per 100 µl in FBS-free M199 containing 0, 0.05, 0.2, 0.4 and 0.6 mM palmitate) were placed in the upper chambers. The lower chamber contained 600 µl of M199 supplemented with 20% FBS and cells were incubated for 16 h at 37°C. Following incubation, cells that remained on the upper surface of the membrane were removed by wiping with a cotton swab, while cells that crossed onto the lower side of the membrane were fixed with 4% paraformaldehyde for 30 min at room temperature and subsequently stained with crystal violet for 20 min at room temperature. The number of cells was counted manually in six random microscope fields by two independent investigators. The experiments were repeated in the presence of 10 µΜ cathepsin inhibitors for 1 h prior to the addition of palmitate, as described above for cell culture.

Total RNA was extracted using TRIzol^® reagent (Sigma-Aldrich; Merck KGaA). cDNA was synthesized from 2 µg total RNA using ReverTra Ace^® qPCR Reverse Transcriptase kit (cat. no. FSQ-101; Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's protocol. qPCR was performed using the SYBR-Green Master mix (cat. no. QPK-212; Toyobo Co., Ltd.) in an ABI 7500HT Real-Time PCR machine (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions for PCR were 95°C for 60 sec, followed by 40 cycles at 95°C for 15 sec, 61°C for 15 sec and 72°C for 45 sec. The relative expression level was determined by the 2^−∆∆Cq method and normalized against GAPDH (15). The primer sequences used were as follows: Forward, 5′-CGAATCATTGAAGATCCGAGTG-3′ and reverse, 5′-ATGGCTTAGAGCCCAATTATGT-3′ for cathepsin L; forward, 5-TAT TGC CTG ATT CTG TGG ACT G-3 and reverse, 5-TGA TGT ACTG GAA AGC CGT TGT-3 for cathepsin S; forward, 5′-GCAGATCGTAGCTGGGGTGAACT-3′ and reverse, 5′-AAGCAAGAAGGAAGGAGGGAGGG-3′ for cystatin C; and forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′ for GAPDH.

Cells were lysed in ice-cold radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) supplemented with 1 mM phenomethylsulfonyl fluoride (Thermo Fisher Scientific, Inc.). The proteins (20 µg per lane) were separated on a 12% SDS-PAGE gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrotransferred onto a polyvinylidene difluoride membrane. Following transfer, blocking was performed with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Membranes were probed with specific primary antibodies at 4°C overnight and horseradish peroxidase (HRP)-conjugated anti-rabbit/mouse immunoglobulin secondary antibodies (1:5,000; cat. nos. SC-2004 and SC-2005; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The primary antibodies used were mouse anti-cathepsin L (1:1,000; cat. no. ab6314; Abcam, Cambridge, UK), rabbit anti-cathepsin S (1:5,000; cat. no. ab50400; Abcam), rabbit anti-cystatin C (1:500; cat. no. ab33487; Abcam) and mouse anti-tubulin (1:1,000; cat. no. AT819; Beyotime Institute of Biotechnology, Haimen, China). Enhanced chemiluminescence was performed using the Pro-light HRP Chemiluminescence kit according to the instructions of the manufacturer (Tiangen Biotech Co., Ltd., Beijing, China). Membranes were scanned and visualized by an LAS3000 system (LAS3000 mini ImageReader, Fuji Photo Film Co., Ltd., Japan).

Cells were lysed and treated with reaction buffer that was included in the kits used for cathepsin L (cat. no. ab65306; Abcam) or cathepsin S (cat. no. ab65307; Abcam), and 10 mM fluorogenic Ac-FR-amino-4-trifluoromethyl coumarin (AFC) substrate, which is the preferred cathepsin L substrate (cat. no. ab65306; Abcam) or 10 mM Ac-VVR-AFC, which is the preferred cathepsin S substrate (cat. no. ab65307; Abcam) according to the manufacturer's protocol. Fluorescence was measured using SpectraMax M5 fluorometer, at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Data are expressed as the mean ± standard deviation of at least three independent experiments. Multiple group comparisons were performed by one-way analysis of variance followed by the least significant difference post hoc test. All tests analyzed were two-sided. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were conducted using SPSS software version 11.5 (SPSS, Inc., Chicago, IL, USA).

HUVECs express the endothelial marker protein CD31 as previously described (16), and as presented in Fig. 1A. To examine the effect of palmitate on HUVEC proliferation, HUVECs were incubated with different concentrations of palmitate and it was observed that palmitate decreased HUVEC proliferation in a dose-dependent manner (Fig. 1B). High levels of palmitate (0.6 mM) inhibited the proliferation rate significantly by 44±7% (P<0.05 vs. 0 mM), whereas the lowest concentration (0.05 mM) had no significant effect (P=0.09 vs. 0 mM). These results are consistent with a previous report (17). To determine whether cathepsin is associated with HUVEC proliferation in the presence of a high concentration of FFA, cysteine protease inhibitors were added to the concentrations of palmitate. It was observed that treatment with the cathepsin L inhibitor Z-FF-FMK (Fig. 1C) and the cathepsin S inhibitor Z-FL-COCHO (Fig. 1D) did not result in significant alterations of the growth rate (P>0.05), suggesting palmitate reduced HUVEC proliferation via a cathepsin-independent pathway.

Palmitate may trigger apoptotic pathways in HUVECs (17); therefore, the present study examined whether cathepsin regulated palmitate-induced HUVEC apoptosis (Fig. 2A). As presented in Fig. 2B, cathepsin L and S inhibitors exhibited the ability to reduce the early apoptosis of HUVECs induced by palmitate by almost 50%, suggesting a key cathepsin-dependent pathway in palmitate-induced apoptosis. Furthermore, cathepsin L inhibitor Z-FF-FMK exhibited the ability to reduce early apoptosis more effectively compared with the cathepsin S inhibitor Z-FL-COCHO. However, late apoptosis appeared to be unaffected by cathepsin inhibitors (Fig. 2C). These results suggested that cathepsin L and S are associated with cell apoptosis as the cathepsin inhibitors protected cells from high FFA level-induced apoptosis.

Previous reports have demonstrated that cathepsin L is required for endothelial progenitor cell invasion and neovascularization (3,18); however, whether other cathepsins may influence cell invasion and whether the effects of decreased invasion result from increased FFA levels remains to be elucidated. The present study firstly assessed the effect of palmitate on cell invasion ability using a Transwell assay. It was observed that 0.4 and 0.6 mM palmitate reduced endothelial cell invasion by 88 and 94%, respectively, compared with untreated cells (Fig. 3), indicating that palmitate inhibits cell invasion in a dose-dependent manner. Furthermore, cathepsin S and L inhibitors significantly reduced the cell migration by almost 2-fold, even in the presence of a high level of palmitate (Fig. 3). These findings indicated that cathepsins contribute to cell migration and invasion and palmitate is a potential factor that may reduce cathepsin-mediated cell invasion.

To investigate the underlying mechanism of cathepsins L and S reduction in response to high FFA levels, the present study measured the effect of high palmitate levels on the mRNA and protein expression levels, and activities of cathepsin L and S in HUVECs. The protease activity experiment demonstrated that cells incubated in palmitate for 24 h presented a markedly attenuated cathepsin L activity (P<0.05; Fig. 4A) compared with those cells cultured in medium without palmitate. The greatest palmitate level decreased the activity of cathepsin L by almost 3.6 fold (Fig. 4A). In addition, the high palmitate level attenuated cathepsin S activity; however, this alteration was not significant (Fig. 4A). To further analyze the effect of high palmitate levels on protease expression, the present study detected the mRNA expression levels of cathepsin L, S and their natural inhibitor cystatin C, and observed that palmitate an increased expression levels in a dose-dependent manner (Fig. 4B and C), suggesting a regulatory interaction between palmitate and cathepsins or their endogenous inhibitor. Consistent with the mRNA expression, cathepsin L protein level was upregulated by high palmitate levels at 0.4 mM but was downregulated by 0.6 mM palmitate (Fig. 4D and E), thus, high palmitate levels (>0.4 mM) may have a role in inhibiting cathepsin L activity. However, the protein expression levels of cathepsin S and cystatin C appeared to vary only slightly with different palmitate concentrations, the differences between the mRNA and protein results may be a result of posttranscriptional modifications.