|Year : 2014 | Volume
| Issue : 3 | Page : 603-610
Effect of sarcosine on endothelial function relevant to angiogenesis
Peruman R Sudhakaran1, Sheela Binu2, Sasikumar J Soumya2
1 Department of Computational Biology and Bioinformatics; Department of Biochemistry, University of Kerala, Thiruvananthapuram, Kerala, India
2 Department of Biochemistry, University of Kerala, Thiruvananthapuram, Kerala, India
|Date of Web Publication||14-Oct-2014|
Peruman R Sudhakaran
Department of Computational Biology and Bioinformatics, Inter University Centre of Excellence in Bioinformatics, University of Kerala, Kariavattom, Thiruvananthapuram, Kerala - 695 581
Source of Support: KSCSTE, Government of Kerala, India; DST, Government of India; UGC, New Delhi, India, Conflict of Interest: None
Aim of Study: Endothelial cells (ECs) respond to changes in metabolic status and switch over to angiogenic phenotype. There are several metabolites known to mediate this transition; however, the effect of sarcosine that accumulates in invasive prostate cancer is not known. The objective of the study was to examine whether sarcosine influences EC function and affects angiogenesis.
Materials and Methods: The effect of sarcosine was studied using different model systems including chick chorioallantoic membrane (CAM), rat aortic rings in culture, and human umbilical vein ECs (HUVECs) in culture. The statistical significance of difference was analyzed by one-way analysis of variance (ANOVA) and Student's t-test using GraphPad 5 software.
Results: Increased vascularization in CAM, increased endothelial sprouting in rat aortic rings in culture, and increased expression of CD31 and E-selectin suggested a possible angiogenic effect of sarcosine. Sarcosine modulated expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). In ECs in culture LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt pathway and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) reversed the effect of sarcosine. Further, sarcosine induced upregulation and activation of Akt in HUVECs.
Conclusion: These results suggest that sarcosine modulates EC function relevant to angiogenesis through modulation of PI3K/Akt/mTOR pathway.
Keywords: Angiogenesis, aortic ring assay, CAM assay, HUVECs, Sarcosine
|How to cite this article:|
Sudhakaran PR, Binu S, Soumya SJ. Effect of sarcosine on endothelial function relevant to angiogenesis. J Can Res Ther 2014;10:603-10
| > Introduction|| |
Angiogenesis is the process of formation of blood capillaries from preexisting vessels,  which occurs during various physiological conditions including wound healing and pathophysiological conditions such as tumor progression. It is regulated by various angiogenic factors such as vascular endothelial growth factor (VEGF).  Apart from external stimuli, angiogenesis is also modulated by the microenvironmental changes within the cells. For instance during wound healing and tumor progression where angiogenesis is critical, the level of lactate in tissue rises up to 10-15 mM as compared to 1.8-2 mM under physiological conditions. ,, Cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria like most normal cells.  During such conditions of ischemia and hypoxia, cells switch over to anaerobic metabolism with concomitant accumulation of certain metabolites.  Some of the metabolites that accumulate in tumors have been shown to be proangiogenic. Lactate has been shown to induce proangiogenic effect in endothelial cells (ECs) by modulating the angiogenic potential of VEGF. 
Level of sarcosine and N-methyl glycine was reported to be elevated in invasive prostate cancer cell lines relative to benign prostate epithelial cells and can be detected noninvasively in urine. Sreekumar et al., hypothesized that prevalence of sarcosine which is derived from glycine by glycine N-methyltransferase or dimethylglycine dehydrogenase, increases with severity of prostate cancer. As prostate cancer pogresses towards metastatic disease, amino acid metabolism along with nitrogen breakdown pathway also increases.  Progression of cancer towards metastatic condition is facilitated by angiogenesis.  Neovascularization mediated by angiogenesis promotes tumor progression by allowing it to maintain its growth advantage and to undergo metastatic spreading. The microvessel density in tumors has been correlated with their metastases. , It is not known whether sarcosine affects angiogenesis. Therefore, the objective of the study was to examine whether sarcosine influences EC function and affects angiogenesis. The results presented here show that sarcosine accumulation affects EC activities relevant in angiogenesis.
| > Materials and methods|| |
MCDB-131 medium, antibiotic-antimycotic solution, o-phenylenediamine dihydrochloride, Tris, protease inhibitor cocktail, monoclonal antibodies against VEGF, fibroblast growth factor (FGF), E-sel, CD31, Akt, mammalian target of rapamycin (mTOR), phoshoP38 mitogen-activated protein kinase (MAPK) and phosphoserine, and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from M/s Sigma Aldrich Co. Tissue culture plastic wares were purchased from NUNC A/S, Roskilde, Denmark. Chick embryos were obtained from regional poultry farm, Trivandrum. All other reagents were of extra pure quality from Merck Ltd, Mumbai, India.
Isolation and culture of human umbilical vein ECs
HUVECs were isolated by collagenase perfusion of the umbilical vein.  The viability of isolated cells was determined by Trypan blue exclusion. Cells in serum free MCDB-131 medium were seeded in plastic Petri dish More Detailses, allowed to attach overnight, unattached cells were removed, and attached cells were maintained in culture at 37°C in 95% air and 5% CO 2 atmosphere in a Sanyo carbon dioxide incubator, Japan. Morphological changes were examined microphotographically. Experiments were carried out with approval of the Institutional Ethics Committee.
Aortic ring assay for angiogenesis
Aortic ring assay was carried out by the method of Nicosia and Ottinetti  as described before.  Aortic rings were placed in 35 mm culture plates, supplied with MCDB-131 medium with and without sarcosine and incubated at 37°C in 95% air and 5% CO 2 atmosphere in a carbon dioxide incubator. The morphological changes were examined under a Leica microscope. The sprouts were quantitated using QWIN software (Leica, Germany).
Chick chorioallantoic membrane assay
CAM assay was performed as described earlier.  Briefly, fertilized chick eggs were incubated for 4 days at 37°C and a relative humidity of 80%. After incubation, the eggs were opened on the air sac side, shells were removed carefully with forceps, and the samples soaked in filter disc were applied on to the CAM. The cavity was covered with parafilm and the eggs were incubated at 37°C and a relative humidity of 80% till 12 th day. The CAMs were photographed after 10 th and 12 th day of incubation and estimated hemoglobin in the CAM using Drabkin's reagent as a measure of vessel density.
Enzyme linked immunosorbent assay
Amounts of E-sel, CD31, VEGF, and FGF were quantitated by ELISA  using HRP-conjugated secondary antibody. o-Phenylene diamine dihydrochloride was used as substrate. Color intensity at 495 nm was read in a multiwell microplate reader (Thermo Multiskan Spectrum, USA) and results were expressed as optical density (OD) units/mg protein. Protein was estimated by the method of Lowry et al. 
Western blot analysis
Production of CD31, VEGF, Akt, mTOR, and phoshoP38 MAPK; and activation of Akt and mTOR were determined by Western blot analysis.  Proteins were separated in a 10% polyacrylamide gel and transferred onto nitrocellulose membrane. Western blot analysis was performed with antihuman CD31, antihuman VEGF, anti-Akt, anti-mTOR, and anti-phosphoserine antibodies at a dilution of 1:1,000. Cell lysate prepared in radio-immunoprecipitation assay (RIPA) lysis buffer was used for Western blot analysis of Akt and mTOR. The activation of Akt and mTOR were studied by probing immnuoprecipitated Akt and mTOR blotted on to nitrocellulose membrane using anti-phosphoserine antibody. The membrane was then incubated with secondary anti-mouse immunoglobulin (Ig) G and anti-rabbit IgG conjugated to HRP (dilution of 1:2,000), respectively. The bands were detected by staining with 3,3'-diaminobenzidine and relative intensity of bands was quantitated using BioRad Quantity One version 4.5 software in a BioRad Gel Doc, USA.
Results are expressed as mean with standard error of mean. The statistical significance of difference was analyzed by one-way analysis of variance (ANOVA) and Student's t-test using GraphPad 5 software. A value of P < 0.05 was considered significant.
| > Results|| |
Angiogenic effect of sarcosine
The angiogenic effect of sarcosine was studied using rat aortic rings in culture. Aortic rings were maintained in culture supplemented with different concentrations of sarcosine (0.1-1 mM) and monitored endothelial sprouting at regular intervals. There was progressive increase in endothelial sprouting in aortic rings treated with sarcosine with progression of culture, unlike untreated controls. Sprouting was observed in sarcosine treated rings in 24 h; whereas, in untreated control significant sprouting was observed in 72 h. Significantly, more sprouting was observed in aortic rings treated with sarcosine at a concentration of 0.1 mM and above than the untreated control at all the time points tested [Figure 1].
|Figure 1: Angiogenic effect of sarcosine-Aortic Ring assay: Rat aortic rings maintained in MCDB 131 medium were treated with sarcosine (0.1mM, 1mM). Rings maintained in medium without any supplement served as control. The morphological changes were photographed under a microscope every 24 hours (x4) (a). Endothelial sprouts were seen in sarcosine treated rings. The sprouts were quantitated using QWIN software (Leica) and expressed in area in ìm2 (b). The values given are average of five experiments ± SEM.*significant when compared to control (P < 0.05)|
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The proangiogenic effect of sarcosine was also observed in CAM. There was progressive increase in vascularity in CAM with time. Treatment of CAM with sarcosine at a concentration of 0.1 mM and above induced a significant increase in vascular density by 10 th day when compared to untreated controls. Hemoglobin level, estimated as a measure of vessel density was maximum in CAMs treated with 1 mM concentration of sarcosine for 10 days. Though vascularity in CAMs treated with 0.1 mM sarcosine for 12 days was not significantly different from that of untreated control, sarcosine at higher concentrations produced significantly higher vascularity during this time interval [Figure 2]. Sarcosine at a lower concentration (0.01 mM) did not produce any increase in vascular density in CAM or sprouting in aortic rings when compared to untreated controls during the time points tested (data not shown).
|Figure 2: Angiogenic effect of sarcosine - CAM assay: Four day old chick embryos were treated with sarcosine (0.1mM, 1mM) and the embryos without any supplement served as control. CAMs were isolated after 10, 12 days and photographs were taken (a). The level of hemoglobin as a measure of the vessel density was determined (b). The values given are average of five experiments±SEM.*significant when compared to control (P < 0.05). No symbol-not signifi cant|
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Effect of sarcosine in ECs
The effect of sarcosine on angiogenesis was examined further using ECs in culture. HUVECs in culture were treated with different concentrations (10 μM-1 mM) of sarcosine. The production of biochemical markers such as CD31 and E-Sel was monitored [Figure 3]. There was significant increase in the levels of cell associated CD31 and E-sel in cells treated with sarcosine. While a significant increase in CD31 was produced by sarcosine at a concentration of 10 μM and above, significant increase in E-sel was produced by sarcosine at concentrations 0.1 mM and above [Figure 3]. To examine whether the effect of sarcosine was specific and not a rather general effect, the cells were treated with L-alanine (1 mM), an isomer of sarcosine. Treatment of cells in culture with L-alanine (1 mM) did not produce any significant difference in the levels of CD31 and E-sel when compared to untreated controls excluding a possible nonspecific effect of sarcosine. The effect of sarcosine on cell-associated CD31 was also examined by maintaining the cells in culture for different time periods in presence of 1 mM sarcosine [Figure 4]. There was progressive increase in the level of cell associated CD31 with progression of culture; its level was significantly high in cultures treated with sarcosine at the time intervals tested, though the rate of production was not significantly different from controls. The increase in the level of CD31 was confirmed by immunoblot analysis.
|Figure 3: Effect of sarcosine on production of CD31, E-selectin, VEGF and FGF by endothelial cells: HUVECs were maintained in culture supplemented with 10μM-1mM sarcosine, 1mM alanine in MCDB 131 medium for 24 hours. Cultures without supplement served as control. The levels of CD 31 (a), E-sel (b), VEGF (c) and FGF (d) were estimated by ELISA using anti CD 31, anti E-sel, anti VEGF and anti FGF respectively. The values given are average of five experiments ± SEM.*significant when compared to control (P < 0.05). No symbol-not significant|
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|Figure 4: Time dependant effect of sarcosine on CD31 and VEGF production by endothelial cells: HUVECs were maintained in culture supplemented with 1mM sarcosine in MCDB 131 for 24 hours and 48 hours. Cultures without supplement served as control. The levels of CD 31 (a) and VEGF (b) were estimated by ELISA using anti CD 31and anti VEGF respectively. Cell layer and medium from cultures maintained for 48 hrs were also subjected immunoblot analysis for CD31 (c) and VEGF (d) respectively. â-actin was immunoblotted as internal control. Intensity of immunoblotted bands were measured as described in the text. The values given are average of five experiments±SEM.*significant when compared to control (P < 0 .05)|
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The effect of sarcosine on ECs was further studied by analyzing the production of angiogenic factors such as VEGF and FGF. Though it produced significant increase in the levels of both VEGF and FGF at 0.1 mM concentration and above, there was no increase in cells treated with 10 μM sarcosine [Figure 3]. Treatment of cells with alanine did not produce any significant change in the levels of VEGF and FGF [Figure 3]. The effect of sarcosine on angiogenic growth factors was also studied by maintaining the cells in culture for different time intervals in presence of 1 mM sarcosine. There was progressive increase in the levels of VEGF in the medium with progression of culture; its level was significantly high in cultures treated with sarcosine at the time intervals tested [Figure 4]. About 1.3-1.5-fold increase in the level of VEGF was observed in HUVECs treated with 1 mM sarcosine [Figure 4]. Western blot analysis confirmed the upregulation of VEGF in cells in culture treated with sarcosine. These results showed that lower concentrations of sarcosine did not induce angiogenic response in ECs and the effect was sarcosine-specific.
Intracellular effect of sarcosine
To study the mechanism of action of sarcosine, effect of sarcosine on certain intracellular signaling pathways relevant in angiogenesis was studied. For this, the production of CD31 and VEGF was studied in HUVECs treated with LY294002 (10 μM), an inhibitor of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and rapamycin (250 nM), an inhibitor of mTOR which is a downstream target of Akt. The levels of these proteins were significantly reduced in cells treated with both sarcosine and LY 294002 [Figure 5] when compared to those of cells treated with sarcosine suggesting that the effect of sarcosine is reversed by LY 294002. The level of VEGF was significantly reduced in cells treated with both sarcosine and rapamycin [Figure 5]. Involvement of Akt and mTOR was further tested by analyzing their activation through phosphorylation. Immunoblot analysis showed that phosphorylation of Akt and mTOR increased significantly in cells treated with sarcosine compared to untreated controls [Figure 6]. To examine whether effect of sarcosine was specific, phosphorylation of p38 MAPK, another signaling protein was studied. However, treatment with sarcosine did not produce any significant change in phosphorylated p38 MAPK.
|Figure 5: Reversal of effect of sarcosine on CD31 and VEGF production in HUVECs by LY294002 and rapamycin: HUVECs were maintained in culture supplemented with sarcosine (1mM), sarcosine (1mM)+LY294002(10μM) and sarcosine (1mM)+rapamycin(250nM) for 48 hours. PBS treated cells served as the control. The level of cell associated CD 31 (a) and levels of VEGF (b) secreted into medium were estimated by western blot analysis. The values given are average of five experiments ± SEM.*significant when compared to control (p < 0.05). #significant compared to sarcosine treated group|
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|Figure 6: Effect of sarcosine on Akt and mTOR in HUVECs: Immunoblot analysis: HUVECs were maintained in culture supplemented with sarcosine (1mM) for 48 hours as indicated in legends to fig5. The levels of Akt and pAkt (a), mTOR and pmTOR (b) and â actin as internal control were analyzed by immunoblotting as described in the text and the corresponding band intensities were plotted. The level of phosphop38MAPK (c) in cells was also analysed by immunoblotting. The values given are average of five experiments±SEM.*significant when compared to control (p < 0.05). No symbol-not significant|
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| > Discussion|| |
Accumulation of certain metabolites has been reported to facilitate tumor progression by angiogenesis. Hypoxic changes induce angiogenic phenotype in ECs with accumulation of metabolites. Though sarcosine has been reported to be increased in prostate tumor,  little is known about its effects on ECs and on angiogenesis. Results presented here show that sarcosine promotes angiogenesis. Evidence in support to this are the following; (a) increased vasculature in CAM and increased endothelial sprouting in aortic rings treated with sarcosine and (b) increased expression of CD31 and E-sel by sarcosine in HUVECs. The adhesive interactions among ECs are important for the formation of new blood vessels and hence are critical for angiogenesis. PECAM-1/CD31 is a cell adhesion molecule that mediates cell-cell interaction during angiogenesis. Inhibiting CD31 blocks tube formation and neovascularization.  E-selectin also plays an important role in the angiogenesis induced by VEGF.
Angiogenic response is often mediated by angiogenic growth factors particularly VEGF. VEGF produced by ECs, hematopoietic cells, and cancer cells regulate blood and lymphatic vessel development. VEGF is produced in response to hypoxia, stimulation by growth factors such as transforming growth factors, platelet-derived growth factor, etc.  FGF, another angiogenic growth factor, binds to its specific receptor FGFR and triggers an intracellular signal cascade leading to multiple biological responses, including EC proliferation, migration, differentiation, and angiogenesis.  The results presented here show that the angiogenic effect of sarcosine involves the modulation of these growth factors. ELISA and Western blot analysis confirmed that sarcosine caused the upregulation of these growth factors in HUVECs suggesting that sarcosine promotes angiogenesis by increasing the production of angiogenic growth factors by the ECs. However, sarcosine seems very mild in vitro on VEGF production.
In order to examine how sarcosine affects production of angiogenic growth factors, the intracellular effect of sarcosine was studied. The PI3K/Akt pathway regulates multiple critical steps in angiogenesis including EC survival, migration, and capillary-like structure formation.  Sarcosine appears to exert its effect intracellularly by acting on PI3K/Akt pathway. This was evident from the significant downregulation of CD31 and VEGF in cells treated with LY294002, an inhibitor of PI3K/Akt pathway. Sarcosine induced significant upregulation and activation of Akt in HUVECs in vitro. This was evident from the following observations; (a) increase in the level of Akt in HUVECs maintained in sarcosine supplemented medium as compared to untreated control (data not shown) and (b) increase in the levels of serine phosphorylation of Akt in sarcosine supplemented HUVECs as compared to untreated control cells. The major signaling pathways that are altered during advanced prostate cancer are Akt/mTOR and MAPK pathways. Kinkade et al., reported that a combinatorial inhibition of Akt/mTOR and extracellular signal-regulated kinases (ERK)/MAPK signaling is an effective strategy for inhibiting prostate tumorogenicity in vivo.  Constitutive PI3K/Akt signaling contributes to the progression of prostate cancer from organ-confined condition to highly invasive condition.  Our result on the increased activity of PI3K/Akt in HUVECs by sarcosine is important as it is reported to increase in prostate cancers. 
Since the expansion of AKT-driven prostate epithelial cells requires mTOR-dependent survival signaling, we examined the possibility of mTOR being a downstream target of Akt in sarcosine-mediated signaling cascade. The role of mTOR was studied using rapamycin, a specific inhibitor of mTOR. There was significant downregulation in the level of CD31 and VEGF in HUVECs treated with sarcosine along with rapamycin. The PI3K/Akt/mTOR pathway has been implicated in hypoxic response by HIF in transformed cells. , mTOR is a possible downstream target of Akt in inducing VEGF expression and hence angiogenesis. There are also reports suggesting the role of mTOR signaling in certain metabolite induced angiogenesis. 15S-Hydroxyeicosatetraenoic acid, a lipoxygenase metabolite of arachidonic acid, induces angiogenesis through PI3K/Akt/mTOR/S6K1 signaling.  The role of mTOR on sarcosine-induced signaling pathway was further confirmed by increased serine phosphorylation of mTOR in HUVECs treated with sarcosine. mTOR modulates the expression of several transcription factors and P70S6K, 4E-BP1, and HIF are some of the possible downstream targets of mTOR signaling pathway.  Since sarcosine modulates mTOR activity in EC, it is possible that sarcosine could alter the expression of mTOR responsive genes. Sarcosine may modulate regulation of HIF1α, thereby inducing increased production of VEGF.
Sarcosine is a normal physiological metabolite and the concentration of sarcosine in blood serum of normal human subjects is reported to be 1.59 ± 1.08 μM/L.  However, sarcosine level increased several fold in tissue and urine of prostate cancer patients. , The angiogenic effect of sarcosine was observed at concentrations 0.1 mM and above in the model systems tested in our study; no major effect was observed at lower concentrations. In vitro studies on prostate cancer cell motility and invasiveness showed that sarcosine at concentrations of 10-50 μM range produced significant stimulatory effect.  It therefore appears that the angiogenic effect of sarcosine observed here may be more relevant in pathological conditions and may not be significant under normal physiological conditions where the sarcosine level is very low. Further, these findings are also important in the context of the reported pharmacological effects of sarcosine in treatment of schizophrenia. 
A schematic representation of the effect of accumulation of sarcosine is given in [Figure 7]. Though the precise molecular target of sarcosine could not be identified, it appears that sarcosine accumulation in ECs results in activation of PI3K which in turn may activate Akt and mTOR leading to modulation of a downstream target to induce production of angiogenic factors and hence angiogenesis.
|Figure 7: Mechanism of angiogenic effect of sarcosine: Sarcosine when accumulates in endothelial cells activates PI3K, Akt and mTOR. mTOR modulates a downstream target to induce VEGF production and angiogenesis|
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| > Acknowledgment|| |
The financial assistance to Binu S in the form of fellowship from Kerala State Council for Science Technology and Environment, Government of Kerala and to Soumya SJ from UGC in the form of RFSMS fellowship, New Delhi is gratefully acknowledged. We are thankful to the doctors and nursing staffs of Gowreesha Hospital and Anadiyil Hospital, Thiruvananthapuram for supplying umbilical cord for this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]