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Palmitate impairs angiogenesis via suppression of cathepsin activity

Angiogenesis requires the interaction of multiple variable factors to promote endothelial cell adhesion, migration and survival. Palmitate, a free fatty acid, exhibits an anti-angiogenic effect via interference with endothelial cell function, whereas cysteine proteases are important in protein turnover and are termed positive modulators of neovascularization. However, the association between these two factors regarding the regulation of human endothelial cell function remains to be elucidated. By using cell counting kit-8, the Transwell method and an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit, the present study reported that high levels of palmitate result in a significant decrease in endothelial cell proliferation and invasion, and induced cell apoptosis; cathepsin L and S inhibitors may suppress palmitate-induced apoptosis. Conversely, the results of the cathepsin L and S activity assay and reverse-transcription-quantitative polymerase chain reaction indicated that palmitate inhibited 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.


Introduction
Angiogenesis is a complex multi-step process that occurs in response to ischemic stimuli. These steps include degradation of the basement membrane, proliferation, apoptosis and invasion of endothelial cells, tube formation, and anastomosis (1,2). Various proteases including the family of matrix metalloproteinases (MMPs), and cysteine and serine proteases contribute to the complex processes of new vessel formation and remodeling. Urbich et al (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 et al (4) demonstrated that cathepsin S was required for microvessel formation. Cysteine proteases have previously been demonstrated to be important in apoptosis and cell survival, separate from their role in proteolysis of extracellular matrix in vascular remodeling (5)(6)(7); 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 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, Cell proliferation assay. 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.
Cell apoptosis assay. 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 1x10 6 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.
In vitro invasion. 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 (1x10 5 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.
Cathepsins L and S activity assay. 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.
Statistical analysis. 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).

Results
Effect of palmitate and cysteine protease on HUVEC proliferation. 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.
Cysteine protease inhibitors attenuate palmitate-induced HUVEC apoptosis. 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.

Palmitate inhibits cathepsin-mediated cell invasion.
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.
Palmitate suppresses the activity of cathepsins L and S. 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.

Discussion
The present study demonstrated that exposure of cultured HUVECs to palmitate led to impairment of cell proliferation and invasion, and increased cell apoptosis, and that the function of palmitate is associated with cathepsin L and S. Further experiments demonstrated that palmitate inhibited the activity of cathepsin L and S in HUVECs.
The effects of FFA on cell proliferation, apoptosis and invasion have previously been investigated; Listenberger et al (10) observed that saturated fatty acids increase nicotinamide adenine dinucleotide phosphate oxidase-generated oxidant stress, nuclear factor-kB activation and ceramide accumulation. The latter, which may be synthesized de novo from saturated fatty acids and the amino acid serine, has been implicated in apoptosis and oxidative stress modulation (19)(20)(21). Consistent with these reports, high concentrations of saturated fatty acids may trigger apoptosis and inhibit cell cycle progression in cultured human endothelial cell monolayers (22)(23)(24). In addition, trophoblast cells treated with long-chain fatty acids revealed increased lipid droplet deposition, severe mitochondrial damage and significantly decreased invasion (25). The present study demonstrated that the FFA palmitate exhibits similar effects on HUVECs.
Cysteine proteases contribute to angiogenesis under pathophysiological conditions. Cathepsins were originally identified as members of the cysteine protease family localized in lysosomes. However, previous data revealed unexpected roles for cathepsins in pathological conditions including cell survival, metabolic disorder and atherosclerosis-based cardiovascular disease (26)(27)(28)(29)(30)(31). However, the definite underlying molecular mechanism remains to be fully elucidated. A variety of cathepsins (B, D, L, S and K) have been demonstrated to cleave the pro-apoptotic B-cell lymphoma-2 (Bcl-2) family member BH3 interacting domain death agonist (Bid) into its potent pro-apoptotic tBid fragment in vitro (32,33). Translocation of these pro-apoptotic fragments to the mitochondrial outer membrane may induce the release of apoptogenic factors including cytochrome c and subsequent activation of downstream caspases. In addition to Bcl-2 family members, caspases may be appropriate candidates as cathepsin substrates (34)(35)(36)(37)(38)(39). It was therefore hypothesized that cysteine proteases contribute to the regulation of apoptotic signaling mechanisms. Consistent with this, the present study observed that cathepsin inhibitors protected cells from high FFA level-induced apoptosis. In addition, it has previously been demonstrated that intracellular and extracellular cathepsin S, L and K activities were reduced in non-invasive compared with highly invasive cells (40), and downregulation of cathepsins L and S led to defective endothelial cell invasion and suppression of proliferation (41,42), indicating that cathepsins are important for angiogenesis. Therefore, cathepsins S and L are required for endothelial progenitor cell-induced neovascularization in response to ischemic stress (3,43).
In conclusion, the results of the present study, in conjunction with previous findings, support the anti-angiogenic action of FFA. The results suggested that the cathepsin-dependent effects should be considered when developing novel therapies during human endothelial cell angiogenesis. These findings suggested a novel approach to pharmacological and genetic rescue strategies in FFA-mediated angiogenesis.