Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII−/− mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE−/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE−/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE−/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.


Introduction
Arginase hydrolyzes L-arginine to urea and L-ornithine, a precursor for polyamines, including spermine, spermidine, and putrescine. Two isoforms of arginase are found in a variety of cells and may be expressed in species-and tissue-specific manners [1][2][3][4][5] . Arginase I (ArgI) is found in the cytosol, and arginase II (ArgII) is targeted to the mitochondria. Although ArgI can stimulate proliferation through a polyamine-dependent mechanism in aortic smooth muscle cells 6 , the mechanism underlying the role of ArgII in mitochondrial function is yet unknown. We previously demonstrated that ArgII played an important role in the regulation of mitochondrial Ca 2+ concentration in native low-density lipoprotein (nLDL)-treated hAoSMCs, although we did not define the target protein 7 . Together with the endoplasmic reticulum, the mitochondria play a critical role as a Ca 2+ -handling organelle in cells, and mitochondrial Ca 2+ influx from the cytosol affects cellular physiology and pathophysiology through contributions to the spatial and temporal profiles of intracellular Ca 2+ signaling.
Calmodulin contains four EF-hand Ca 2+ binding sites and is a major Ca 2+ sensor and regulatory protein. In vascular smooth muscle cells (VSMCs), increased cytosolic concentration of Ca 2+ ([Ca 2+ ]c) activates the multifunctional Ca 2+ /calmodulin-dependent kinase II (CaMKII) 8 . The activated CaMKII provokes vascular smooth muscle cell (VSMC) contraction through interactions with myosin light chain kinase (MLCK), leading to MLC phosphorylation and actin-myosin interaction 9,10 . Furthermore, increased levels of intracellular Ca 2+ may function as a second messenger to activate other signaling molecules [11][12][13] .
Initially, p32 was characterized as a pre-mRNA splicing factor SF-2 associated protein 14 , as hyaluronan-binding protein 1 (HABP1), as receptor for globular head domains complement 1q (gC1qR), or as complement 1q binding protein (C1qbp) 15 . Moreover, p32 is a conserved eukaryotic protein that exists in multiple subcellular compartments [16][17][18] . However, recent studies have demonstrated that p32 is primarily targeted to the mitochondria because the 73 N-terminal amino acids contain a mitochondrial localization sequence 19 . Mitochondrial p32 protein has been studied previously in cancer development in the context of mitochondrion morphology 20 , the maintenance of oxidative phosphorylation 21 , and ARF-induced apoptosis 22 . However, the primary function of p32 in the mitochondrion is yet unknown.
Although arginase activity in VSMCs provokes cell proliferation in a polyamine-dependent manner 6 , the protein regulated by arginase activity and its function in the mitochondrion are not yet known. To determine the physiological contribution of ArgII activity to vasoconstriction in VSMCs from humans and mice, we investigated how ArgII activity modulates [Ca 2+ ]c and [Ca 2+ ]m through p32. Furthermore, we tested the underlying mechanism of p32-dependent vasoconstriction in deendothelialized vessels.

Isolation of nLDL and cell culture
The nLDL (density 1.019-1.063 g ml −1 ) was prepared from the plasma of normocholesterolemic subjects by differential ultracentrifugation as previously described 7 . Isolated nLDL did not exhibit oxidative modification within 3 weeks compared with the oxidation of commercial nLDL and oxidized LDL from Intracel (Frederick, MD, USA), as determined by thiobarbiturate-reactive substance assays using malondialdehyde as a standard.
Human aortic smooth muscle cells (hAoSMCs) from four different batches were purchased from Lonza, Co. (Allendale, NJ, USA), maintained in medium 231 supplemented with SMGS (Cascade Biologics, Portland, OR, USA) according to the manufacturer's protocol and treated with nLDL (final concentration, 100 µg/ml) after incubation in starvation medium (DMEM, 0.1% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin) for 24 h. mAoSMCs of at least eight different batches were isolated from the thoracic and upper parts of the abdominal aorta of 10-week-old male mice (C57BL/6) as previously described 23 with minor modifications. Briefly, the stripped aorta was prepared from the anesthetized mice and cut into 2-mm pieces, which were treated with type II collagenase (1 mg/ml, Invitrogen, Carlsbad, CA, USA) for 1 hr to remove the endothelial cells. The deendothelialized aortic pieces were incubated with culture medium on gelatin (0.1%)-coated culture dishes for approximately 10 days. The cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 8 mM HEPES, and 2 mM glutamine at 37°C in a humidified 5% CO 2 incubator. Before all experiments, isolated mAoSMCs at passage numbers 1-3 were maintained for 24 h in starvation medium to maintain their contractile phenotype. mAoSMCs were identified by their 'spindle-shaped' pattern and further confirmed by double staining using platelet endothelial cell adhesion molecule-1, a specific marker for endothelial cells, and α-smooth muscle actin, a specific marker for SMCs. All cells were stained with anti-α-smooth muscle actin antibodies. Each experiment was performed in at least two different batches of VSMCs depending on the availability of the cells.

Animals
The study was approved in accordance with the Guide for the Care and Use of Laboratory Animals (Institutional Review Board, Kangwon National University). Studies were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Three pairs of ArgII −/− mice with a C57BL/6 background were gifted generously by Prof. Jaye Chin-Dusting to establish a colony. Mice were anaesthetized by 2.5% isoflurane in O 2 and subjected to the following experiments. Aortic rings were studied from ten 10-weekold male C57BL/6 WT mice fed a normal diet (ND) and ten 10-week-old male ApoE −/− mice (Daehan Biolink, Co.) fed a high-cholesterol diet (HCD, D12108C, Research Diet, Inc., USA) for 8 weeks 1,24 .

Arginase activity
Arginase activity was determined from urea concentration measurements as described previously 4 .
[Ca 2+ ]m and [Ca 2+ ]c measurement using confocal microscopy and flow cytometry Direct assessment of the mitochondrial Ca 2+ content ([Ca 2+ ]m) was performed by an established loading procedure of the cells with calcein acetoxymethyl ester (calcein-AM, Thermo Fisher Scientific, Waltham, MA, USA) and CoCl 2 for mitochondrial localization of calcein fluorescence 25 . Briefly, cells were loaded with 500 nM calcein-AM at 37°C for 30 min in starved media. Calcein-AM generates free fluorescent intracellular calcein upon cleavage of the ester bond. CoCl 2 was then added to the medium to quench the cytosolic calcein fluorescence, while intramitochondrial calcein allowed visualization of the mitochondria as bright fluorescent bodies. Subsequently, the cells were washed free of calcein-AM and CoCl 2 and incubated in Tyrode's modified solution (150 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose). For detection of calcein fluorescence, a 470 nm excitation and a 510 nm emission filter were used. We also tested [Ca 2+ ]m using Rhod-2 AM (Thermo Fisher Scientific, Co.). Briefly, cells were loaded with 2.5 μmol/L Rhod-2 AM at 37°C for 1 h in starved media. Subsequently, the cells were washed free of Rhod-2 AM and incubated in Tyrode's modified solution (NaCl 150 mmol/L, KCl 4 mmol/L, CaCl 2 2 mmol/L, MgCl 2 2 mmol/L, HEPES 10 mmol/L and glucose 10 mmol/L). For detection of Rhod-2 AM fluorescence, 552 nm excitation and 581 nm emission filters were used. MitoTracker green FM (Thermo Fisher Scientific, Co.) was incubated to confirm the mitochondria at 100 nmol/L for 1 h and imaged at 490 nm excitation and 516 nm emission. [Ca 2+ ]c was monitored using Fluo-4 AM (100 nmol/L, 1 h, Thermo Fisher Scientific, Co.) at 494 nm excitation, and emission at 506 nm was detected. Intensity values were normalized according to the initial fluorescence values after subtraction of background using the Metamorph program (Molecular Probe).
[Ca 2+ ]m and [Ca 2+ ]c were also determined using flow cytometry (FACSCalibur). The fluorescence intensity for each sample was determined using CellQuest software. The Ca 2+ level was determined by comparing the fold changes in the fluorescence intensities of treated cells versus control cells.

Mitochondria fractionation
Cells and de-endothelialized aortic segments were homogenized twice in subcellular fractionation buffer (250 mM sucrose, 20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, and protease inhibitors) (Roche, Co., Basel, Switzerland) for 3 min and centrifuged at 1000 × g for 10 min to remove cell debris and unbroken cells. The supernatants were centrifuged at 21,000 × g for 45 min at 4°C. The cytosolic (supernatant) and mitochondrial (precipitate) fractions containing 20 μg proteins were used for subsequent western blotting analyses of p32 protein expression.

Western blot analysis
Cell extract and tissue extract were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed for the densitometry of bands using ImageJ from the National Institutes of Health (NIH) 4 .

Fluorescence detection in aortic tissues
De-endothelialized aortic vessels treated with Cy5conjugated sip32m and scm siRNA for 24 h were fixed in 4% formaldehyde, and frozen sections (5 μm) were used for the detection of Cy5 fluorescence. The slides were examined under a fluorescence microscope (Olympus) linked to a Clara EMCCD digital camera (Andor Technol.) and collected using MetaMorph software.

Polyamine analysis
Intracellular concentrations of L-arginine and polyamine, spermine, spermidine, and putrescine were determined by HPLC using pre-column derivatization with o-phthalaldehyde (OPA) according to a modification of previously published methods 26 . Briefly, L-arginine (100 μM) and polyamine (30 μM/each) were added to cell lysate (0.1 mM) as an internal standard. The samples were extracted on solid-phase extraction cartridges (CBA Bond elute, Varian, Yverdon, Switzerland), and the recovery rate was 87.5 ± 3.9% for L-arginine. Eluates were dried over nitrogen and resuspended in double-distilled water for HPLC analysis. HPLC was performed on a computercontrolled Waters chromatography system (M600E) consisting of an automatic injector (M7725i, Waters Co., Easton, MA, USA) and a fluorescence detector (FP-1520, Jasco Co., Easton, MA, USA). Samples were incubated for 1 min with OPA reagent (5.4 mg/ml OPA in borate buffer, pH 8.4, containing 0.4% 2-mercaptoethanol) before automatic injection to the HPLC. The OPA derivative of L-arginine was separated on a 150 × 4.6 mm-5 μm Zorbax Eclipse (Agilent Technologies, Santa Clara, CA, USA) XDB-C18 column with the fluorescence detector set at Ex 340 nm and Em 450 nm. Samples were eluted from the column with 0.96% citric acid/methanol (70:30), pH 6.8 at a flow rate of 1.5 ml/min.

Vessel reactivity assay
The thoracic aorta was dissected from anesthetized mice (C57BL/6) with isoflurane, and fat and connective tissues were cleaned. The aorta was cut into 1.5-mm rings, and the endothelium was removed gently using a wooden stick. Then, the samples were suspended between two wire stirrups (150 μm) in a myograph (Multi myograph system DMT-620, Aarhus, Denmark) in 10 ml Krebsringer solution (95% O 2 , 5% CO 2 , pH 7.4, 37°C). One stirrup was connected to a three-dimensional micromanipulator and the other to a force transducer. The rings were passively stretched at 10-min intervals in 100 mg increments to reach optimal tone (600 mg). After the arterial rings had been stretched to their optimal resting tone, the contractile response to 100 mM KCl was determined. The response to a maximal dose of KCl was used to normalize the agonist responses across vessel rings. Dose responses to the vasoconstrictor PE (10 −10 -10 −6 M) and NE (10 −9 -10 −5 M) were performed.

Statistical analyses
All data are reported as the mean ± SEM. Statistical significance was determined by the Bonferroni-corrected unpaired t-test for unpaired values. A p value of <0.05 was used as the criterion for statistical significance. Dose responses were analyzed by 2-way analysis of variance (ANOVA) using GraphPad Prism 4.0 Software.

Effect of p32m on vasoconstriction in ApoE −/− mice fed an HCD
In de-endothelialized aortas from WT fed an ND and ApoE −/− mice fed an HCD, sip32m treatment significantly reduced the p32m level (Fig. 7a). With the same treatment, accumulated vasoconstrictor responses to PE and NE were constructed. The attenuated vasoconstriction responses to PE in aortas from ApoE −/− mice fed an HCD compared to WT mice fed an ND were improved with sip32m incubation (Fig. 7b,  no response to an endothelium-dependent vasorelaxant, acetylcholine (Ach, Fig. 7d).

Discussion
In this study, we demonstrated for the first time that the activity of ArgII, a mitochondria-targeting Arg isoform, regulates mitochondrial Ca 2+ transition and CaMKII activation via spermine/p32m-dependent mitochondrial Ca 2+ uptake in VSMCs. Furthermore, downregulation of p32m using siRNA resulted in increased [Ca 2+ ]c and CaMKII/MLC20 phosphorylation and enhanced vasoconstriction in de-endothelialized aortic vessels. In deendothelialized aortas from ApoE −/− mice fed an HCD, both Arg activity and spermine levels increased. Interestingly, p32m was upregulated and [Ca 2+ ]m was augmented in mAoSMCs from atherogenic mice. Consistent with the above data, CaMKII phosphorylation was decreased and vascular tension in response to vasoconstrictors was attenuated in de-endothelialized aortas from ApoE −/− mice fed an HCD. However, incubation with sip32m restored responses to the vasoconstrictors.
Increased Arg activity contributes to the pathophysiology of various disease processes. Vascular endothelial Arg leads to endothelial dysfunction by substrate depletion and contributes to vascular diseases 24,[27][28][29][30] . Consistent with these observations, long-term administration of Larginine in some preclinical studies has been shown to prevent atherosclerosis and myointimal hyperplasia and enhance angiogenesis [31][32][33] . However, in patients with peripheral arterial disease, long-term L-arginine administration resulted in increased levels of ornithine and urea and did not improve vascular reactivity 34 . This discrepancy may be partly explained by our study demonstrating that increased Arg activity provoked the production of spermine, which may induce mitochondrial Ca 2+ uptake via p32m. This phenomenon is a reason for mitochondrial dysfunction. Increased Ca 2+ uptake into mitochondria becomes a pathological stimulus by promoting ROS generation, cytochrome C release, and apoptosis [35][36][37] .
Mitochondrial Ca 2+ transport plays a central role in cellular physiology and pathophysiology. Ca 2+ influx from the cytosol can control the rate of mitochondrial energy production through activation of pyruvate dehydrogenase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase in the Krebs cycle 38 and through regulation of Fig. 4 Spermine is a primary activator of mitochondrial Ca 2+ uptake through mitochondrial p32. hAoSMCs were treated with Cy5-conjugated sip32 and scm siRNA for 24 h and stimulated with polyamine, putrescine, spermidine, and spermine (each 100 μM, 3 h). [Ca 2+ ]m levels were imaged by CoCl 2 -quenched calcein-AM fluorescence, and the translocation of siRNA into cells was confirmed by Cy5 imaging (a, upper). Spermine treatment induced an increase in [Ca 2+ ]m that prevented incubation of sip32 (a, lower, * vs. untreated, P < 0.05). n = 3 independent experiments and 10-12 different images were obtained in each experiment. b Spermine decreased CaMKII phosphorylation in nLDL-treated hAoSMCs (* vs. untreated, P < 0.05). Incubation of sip32 restored nLDL-and spermine-induced decreases in CaMKII phosphorylation (c), but scm siRNA had no effect on CaMKII phosphorylation (d). All blots are representative of three independent experiments. e Arginase inhibition with ABH (10 μM) reduced intracellular spermine concentration (top, representative HPLC chromatogram, * vs. untreated, P < 0.05). n = 4 experiments FoF1ATPase 39 and adenine nucleotide translocase 40 activity in the electron transport chain. Thus, increased Ca 2+ uptake into the mitochondria in nLDL-stimulated VSMCs appears to be a natural phenomenon, as nLDL primarily serves to supply lipids and is then catabolized through β-oxidation in mitochondria to acetyl-CoA, which is utilized for the production of energy (ATP). Our study showed that nLDL stimulation exerted an increase in [Ca 2+ ]m that was prevented by Arg inhibition (Fig. 1a) and ArgII gene deletion (Fig. 1c). However, [Ca 2+ ]c was decreased by nLDL stimulation that was also recovered by downregulation of Arg activity (Fig. 1b, d) and resulted in activation of a Ca 2+ -sensitive protein kinase, CaMKII (Fig.  2). In this study, we illustrate a mechanism in which mitochondrial dysfunction is an early phenomenon of cardiovascular diseases initiated by hypercholesterolemia. Our findings are consistent with previous demonstrations that the mitochondria decode and shape cellular Ca 2+ signals [40][41][42] by uptake and release of Ca 2+ ions to modulate cell metabolism and cell death 43 . The main transporters contributing to rapid Ca 2+ uptake into the mitochondria are reported 44 ; herein, we present for the first time a role for p32m as a novel mitochondrial Ca 2+ transporter. Indeed, p32m could form a doughnut-shaped trimer with 48 amino acids, aspartic and glutamic acids, distributed on the surface of the trimer. With its acidic surface, p32m has been suggested as a high-capacity divalent cation storage protein modulating mitochondrial cation concentrations 45 . Consistent with this assumption, we confirmed that p32m is involved in Ca 2+ uptake using siRNA (Fig. 3b). Furthermore, p32m-dependent entry of Ca 2+ into the mitochondria was enhanced with spermine treatment (Fig. 4a), and spermine pretreatment attenuated CaMKII phosphorylation (Fig. 4b). These observations were consistent with a previous report that spermine can play an important role in the regulation of the free cytoplasmic Ca 2+ concentration and of the free Ca 2+ concentration in the mitochondrial matrix, while the responsible channel protein was not identified 46 . Increased [Ca 2+ ]m was shown to regulate cell metabolism 38 , and p32m was shown to be a regulator for maintenance of oxidative phosphorylation 21 . In tumor cells, p32m knockdown cells shift their metabolism from oxidative phosphorylation toward glycolysis 21 . Based on these results, we wanted to confirm the function of p32m in Ca 2+ -dependent contraction in VSMCs. As shown in  In VSMCs from ApoE-null mice fed an HCD, we found that the p32m levels were upregulated and [Ca 2+ ]m was augmented ( Fig. 6c and E). Indeed, Arg activity increased in ApoE −/− mice fed an HCD, spermine concentration was high, and CaMKII phosphorylation was significantly attenuated compared to WT mice fed an ND (Fig. 6a, b, and F). Furthermore, the downregulation of increased p32m using sip32m restored attenuated contraction responses (Fig. 7). These results suggested that p32m expression may be regulated by Arg activity because p32m was downregulated in ArgII −/− and upregulated in ApoE −/− . In VSMCs, intracellular Ca 2+ concentration to maintain basal vascular tone is modulated by Ca 2+ release from the intracellular stores in the sarcoplasmic reticulum, and Ca 2+ entry from the extracellular space through plasma membrane Ca 2+ channels. Here, we present a novel mechanism underlying the regulation of [Ca 2+ ]c by inhibiting Ca 2+ uptake into mitochondria in nLDLstimulated VSMCs. Increased [Ca 2+ ]c activates specific protein kinases and phosphatases involved in VSMC contraction and relaxation. Indeed, Ca 2+ /calmodulindependent kinase II (CaMKII) is activated by elevated [Ca 2+ ]c and autophosphorylated to participate in the cell cycle and growth regulation in VSMCs. In addition, CaMKII has been implicated as a regulator of VSMC contraction. Consistent with these observations, ArgII −/− and p32m knockdown demonstrated augmented activation of CaMKII and enhanced vasoconstrictive responses to agonists in de-endothelialized vessels.
In this current study, two major points should be explained. The first is why [Ca 2+ ]c was maintained at higher levels by downregulating Arg activity than that of untreated cells (Fig. 1b), although Arg inhibition reduced the increases in [Ca 2+ ]m in nLDL-stimulated hAoSMCs to untreated levels. A possible reason is L-arginine content. Temporary inhibition of Arg activity definitely elicited a decrease in intracellular spermine amount and an increase in intracellular L-arginine levels 47 . Interestingly, incubation of cells with L-arginine increased intracellular [Ca 2+ ]c levels (data not shown). Therefore, higher [Ca 2+ ]c by transient inhibition of Arg activity (Fig. 1b) may be attributed to increased intracellular L-arginine contents. The second explanation is that [Ca 2+ ]m in mAoSMCs from ArgII −/− mice was maintained at lower levels than that of WT cells and not increased under nLDLstimulated conditions. As shown in Supplementary Fig.  4, p32m levels were clearly downregulated, and PE-dose In conclusion, we demonstrated that ArgII activity, coupled with spermine production, contributes to mitochondrial dysfunction through increased p32mdependent mitochondrial Ca 2+ uptake. In particular, p32m levels were upregulated, and [Ca 2+ ]m levels were increased in VSMCs from ApoE −/− mice. Finally, downregulation of p32m using the siRNA technique augmented CaMKII phosphorylation and agonist-induced vasoconstriction activity. De-endothelialized aortas from WT mice fed an ND and ApoE −/− mice fed an HCD for 8 weeks were incubated with sip32 and scm siRNA. Incubation of sip32 resulted in a decrease in mitochondrial protein levels (a, representative blot from three independent experiments). In a vascular tension assay using deendothelialized aortas with vasoconstrictive agonists, PE (b) and NE (c), the decreased constriction in aortas from ApoE −/− mice fed an HCD was restored with incubation of sip32 (*, P < 0.01, **, P < 0.01). n = 4 mice. All groups showed no responses to Ach (d)