Endothelial Dysfunction Induced by Exposure to High Glucose Conditions

Abstract

Endothelial dysfunction induced by exposure to high glucose conditions is recognized as one of the earliest events in the pathogenesis of diabetes-related vascular complications. Activation of the NLRP3 inflammasome protein complex governs the production of interleukin-1? (IL-1?) and interleukin-18 (IL-18), two major proinflammatory cytokines involved in endothelial dysfunction and other diabetic comorbidities. Sirtuin-1 (SIRT1) is an important protective factor that is essential for activation of NLRP3 and is down-regulated in response to high glucose. Recently, there has been conflicting evidence regarding the potential risk/benefit of amino amide-based local anesthetics. Ropivacaine is considered to have the best safety profile among the commonly used amide local anesthetics, but the extent of its actions remains incompletely understood. In the present study, we used human umbilical vein endothelial cells (HUVECs) exposed to high glucose conditions to explore the effects of ropivacaine on oxidative stress, inflammation, and activation of the NLRP3 inflammasome. The results of real-time PCR and western blot analysis indicate that ropivacaine treatment exerted significant beneficial effects by rescuing high glucose-induced oxidative stress and downregulating production of IL-1? and IL-18 through inhibition of the NLRP3 inflammasome. We also found that ropivacaine could inhibit secretion of high mobility group box 1 (HMGB1) protein and improve cell viability. Importantly, the results of our SIRT1 knockdown experiments show that the inhibitory effects of ropivacaine against activation of the NLRP3 inflammasome are dependent on SIRT1. Taken together, our findings demonstrate the potential of ropivacaine as a safe and effective therapy against diabetic endothelial dysfunction.
Keywords: NLRP3; epithelial dysfunction; ropivacaine; type II diabetes; vascular inflammation; SIRT1; IL-1?; IL-18

Introduction

Cardiovascular disease is the most common complication associated with diabetes and the leading cause of death among type II diabetes patients [1;2]. The etiopathogenesis of diabetes-associated vascular disease is complicated, but endothelial inflammation and dysfunction are recognized as playing major roles in disease development and progression. In particular, the chronic hyperglycemic state resulting from diabetes-associated reduced insulin production leads to oxidative stress and a sustained inflammatory response in endothelial cells, thereby causing loss of function and increased apoptosis [3;4]. High glucose-induced endothelial dysfunction alters the ability of the endothelium to regulate vasodilation and increases inflammation. It has been reported that among other things, high glucose-induced endothelial dysfunction impairs signal transduction, upregulates expression of endothelial constricting factors, and downregulates the release of endothelium-derived relaxing factor. Meanwhile, degradation of endothelial-derived relaxing factor is increased and the sensitivity of vascular smooth muscle cells to this factor is decreased, thereby further promoting vascular constriction [5]. Oxidative stress has also been implicated in endothelial dysfunction. Exposure to high glucose levels increases cellular and mitochondrial production of reactive oxygen species (ROS) and NADPH oxidase 4 (NOX4) [6]. NOX4 regulates production of ROS and suppression of NOX4 has been shown to ameliorate diabetes-associated endothelial dysfunction [7]. Thioredoxin-interacting protein (TxNIP) is an important inhibitor that is upregulated in response to high glucose and works to inhibit the antioxidant thioredoxin, thereby promoting further imbalance of the oxidant/antioxidant ratio. TxNIP is also a mechanism for activating the NLRP3 inflammasome [8].

The nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) is a member of the nucleotide-binding domain and leucine-rich repeat-containing receptor (NLR) family of receptors [9]. The term inflammasome is used to describe a set of multi-protein systems that are activated in response to infection, inflammation, and the auto-immune response. The NLRP3 inflammasome is an important complex involved in high glucose-induced endothelial dysfunction and inflammatory response. The NLRP3 inflammasome is composed of NLRP3, an apoptosis-associated spec (ASC)-like protein, and caspase-1. This inflammasome plays a key role in promoting inflammation by guiding the production and maturation of interleukin (IL)-1? and IL-18, first through inducing expression of pro-IL-1?/pro-IL-18 and then through activation of caspase-1, which cleaves pro-IL-1? and pro-IL-18 into their active cytokine forms, mature IL-1? and IL-18 [10;11]. Inhibition of NLRP3 inflammasome activation has been considered as a promising therapeutic target against the development of types I and II diabetes [12;9] as well as several diabetes-associated complications, including atherosclerosis [13], diabetic cardiomyopathy [14], and diabetic nephropathy [15]. However, the cellular pathways involved in NLRP3 inflammasome activation are poorly understood.

Ropivacaine is one of the most widely used amide-based local anesthetics in both clinical and dental settings. Local anesthetics work by blocking neural signal transduction through inhibition of sodium ion uptake in a limited area of the body and for a limited amount of time [16]. While research has suggested potential cytotoxic and apoptotic effects of amino amide local anesthetics including bupivacaine, ropivacaine, levobupivacaine, and mepivacaine, a recent literature review reported that among them, ropivacaine had the best safety profile in terms of cytotoxicity [17]. Recent research has also demonstrated a potential anti-inflammatory effect of ropivacaine through inhibition of proinflammatory cytokines including IL-1? [18;19]. Interestingly, a recent study involving macrophages showed that ropivacaine could prevent apoptosis, inhibit expression of IL-1?, IL-6, and tumor necrosis factor-?, and suppress activation of the mitogen activated kinase and necrosis factor-?B inflammatory signaling pathways, thereby significantly suppressing inflammation [19]. Moreover, recent evidence shows that ropivacaine may also exert anti-oxidative stress effects by… Sirtuin-1 (SIRT1) is a class III histone deacetylase involved in regulating innate immunity, cell senescence, apoptosis, metabolism, and cell cycle. SIRT1 has been shown to exert a protective effect against endothelial dysfunction by inhibiting premature cell senescence and downregulating expression of plasminogen activator inhibitor 1 and endothelial nitric oxide [20]. Importantly, SIRT1 has also been shown to negatively regulate activation of the NLRP3 inflammasome in endothelial cells [21]. In the present study, we investigated the role of ropivacaine in high glucose-induced endothelial dysfunction with a focus on the NLRP3 inflammasome and SIRT1. Our findings demonstrate that ropivacaine treatment induced dose-dependent protective effects against high glucose in human umbilical vein endothelial cells (HUVECs) by reducing oxidative stress, inhibiting activation of the NLRP3 inflammasome, and reducing production of IL-1? through inhibition of TxNIP. Importantly, we show that these effects are mediated through Sirtuin-1 (SIRT1).

Materials and methods

Results

The effects of ropivacaine treatment on high glucose-induced oxidative stress were determined by measuring generation of ROS and NOX4. A shown in Figure 1, the results of MitoSOX staining reveal that exposure of HUVECs to high glucose resulted in a greater than 3-fold increase in production of ROS, which was reduced to only 1.5-fold baseline by 0.25% and 0.5% ropivacaine in a dose-dependent manner (1, 3.4, 2.1, 1.5). Next, we measured the effects of ropivacaine on high glucose-induced expression of NOX4. The results of real-time PCR and western blot analysis in Figure 2 indicate that high glucose increased expression of NOX4 at both the mRNA and protein levels, which was ameliorated by ropivacaine in a dose-dependent manner (mRNA: 1, 3.1, 2, 1.6; protein: 1, 2.9, 1.8, 1.4). We assessed cell viability upon exposure to high glucose as well as the two doses of ropivacaine (0.25% and 0.5%) by measuring the release of LDH into the cytoplasm. Release of LDH into the culture medium is a determinate of cell toxicity. As shown in Figure 3, exposure to high glucose significantly increased the rate of cell death, which was ameliorated by treatment with ropivacaine in a dose-dependent manner (3%, 35%, 21%, 13%).

Next, we determined the effects of ropivacaine on high glucose-induced release of HMGB1. As shown in Figure 4, the results obtained using a commercial HMGB1 kit revealed that exposure to high glucose conditions significantly increased secretion of HMGB1, which was suppressed by treatment with ropivacaine in a dose-dependent manner (1, 4.6, 3.1, 1.9). TxNIP plays an important role in activation of the NLRP3 inflammasome and production of IL-1?. To determine whether ropivacaine treatment impacted high glucose-induced TxNIP expression, we performed real time PCR and western blot analyses. As shown in Figure 5, the two doses of ropivacaine could ameliorate high glucose-induced expression of TxNIP in a dose-dependent manner at both the mRNA (1, 3.5, 2.2, 1.5) and protein (1, 3.3, 1.9, 1.4) levels. Next, we investigated the effects of ropivacaine on activation of the NLRP3 inflammasome. As demonstrated by the results of western blot analysis in Figure 6, ropivacaine significantly reduced the levels of NLRP3 protein (1, 3.8, 2.4, 1.6), ASC (1, 3.6, 2.2, 1.6), and cleaved caspase 1 (P10) (1, 4.1, 2.5, 1.7) induced by high glucose in a dose-dependent manner. To confirm that ropivacaine-mediated reduced TxNIP expression and NLRP3 inflammasome activation could inhibit production of IL-1?, we measured the effects of ropivacaine on high glucose-induced expression of IL-1? and IL-18. As shown by the results of real time PCR and western blot analysis in Figure 7, the two doses of ropivacaine significantly ameliorated high glucose-induced increased expression of IL-1? (1, 5.6, 3.3, 1.9) and IL-18 (1, 3.7, 2.3, 1.6).

Finally, we investigated the role of SIRT1 in high glucose-induced NLRP3 inflammasome activation. As shown in Figure 8, the results of real time PCR and western blot analyses show that ropivacaine treatment could reverse the reduction in SIRT1 induced by exposure to high glucose in a dose-dependent manner (mRNA: 1, 0.35, 0.63, 0.89; protein: 1, 0.43, 0.66, 0.92). Next, we performed a SIRT1 knockdown experiment to determine whether ropivacaine-mediated inhibition of NLRP3 is mediated through SIRT1. As shown in Figure 9, knockdown of SIRT1 abolished the inhibitory effects of ropivacaine on the expression of NLRP3 (1, 4.1, 1.8, 3.6), ASC (1, 3.5, 1.5, 3.2), and cleaved caspase 1 (P10) (1, 3.5, 2.2, 1.4). Additionally, inhibition of SIRT1 abolished the inhibitory effects of ropivacaine on IL-1? (1, 4.9, 1.8, 4.5) and IL-18 (1, 3.9, 1.7, 3.7).

Discussion

Endothelial inflammation and dysfunction induced by exposure to high glucose play a leading role in diabetes-associated cardiovascular disease by altering the vasoconstrictive function of endothelial cells and creating a proinflammatory environment. Among the factors contributing to high glucose-induced endothelial dysfunction are oxidative stress, increased cell apoptosis, expression of proinflammatory cytokines, and activation of the NLRP3 inflammasome. ROS-mediated activation of TxNIP and subsequent NLRP3 inflammasome activation have been shown to be key events in the pathogenesis of athersclerosis and cardiovascular risk [22]. However, the exact mechanisms driving NLRP3 inflammasome activation and inflammatory response in diabetic endothelial dysfunction remain poorly understood. In the present study, we investigated the effects of the widely used amide local anesthetic ropivacaine in NLRP3 inflammasome activation and inflammation using HUVECs. Our findings indicate that exposure to high glucose induces endothelial dysfunction through ROS- and TxNIP-mediated activation of the NLRP3 inflammasome. However, treatment with ropivacaine significantly inhibits endothelial dysfunction by preventing oxidative stress, increasing cell viability, and suppressing expression of IL-1?, IL-18 and HMGB1 triggered by activation of the NLRP3 inflammasome. Furthermore, these protective effects of ropivacaine appear to be mediated through the SIRT1 pathway.
Endothelial dysfunction, characterized by dysregulation of vascular constriction and relaxation, is one of the earliest events in the development of diabetic vascular disease [23]. Hyperglycemia is well-documented to induce endothelial dysfunction by disrupting the oxidant/antioxidant balance and promoting expression of proinflammatory cytokines including IL-1?, IL-18, and HMGB1 [24-28]. The findings in Figures 1 and 2 indicate that ropivacaine could suppress high glucose-induced increased production of ROS and NOX4, thereby exerting a protective anti-oxidative stress capacity in HUVECs. Previous studies investigating the effects of ropivacaine and other amide local anesthetics on endothelial cell viability have yielded conflicting results [28;29]. Our findings demonstrate that ropivacaine significantly improved diminished endothelial cell viability induced by high glucose (Figure 3).

High mobility group box 1 (HMGB1) is a cellular stress response signaling protein released by immune cells and upon cell death or injury and has been shown to contribute to diabetic endothelial dysfunction [30;31]. A recent study provided evidence that downregulation of HMGB1 may serve as a potential approach for preventing high glucose-induced endothelial dysfunction by inhibiting inflammation and apoptosis [31]. Our findings demonstrate that ropivacaine treatment can significantly inhibit high glucose-induced expression of HMGB1 (Figure 4). TxNIP is an antioxidant-inhibitory protein that plays a key role in activating the NLRP3 inflammasome. Importantly, TxNIP/NLRP3 inflammasome activation has been implicated as an important causative factor in the development of diabetic vascular disease and endothelial dysfunction [32;33]. The results in Figures 5 and 6 indicate that ropivacaine significantly inhibited high glucose-induced activation of the NLRP3 inflammasome complex by suppressing activation of TxNIP. To our knowledge, this is the first study to demonstrate the effects of ropivacaine on activation of TxNIP as well as NLRP3 inflammasome. Production of the proinflammatory cytokines IL-1? and IL-18, as well as their precursors, is orchestrated by the NLRP3 inflammasome. Endothelial release of IL-1? induced by high glucose has been suggested as the origin of upregulated IL-1? associated with diabetic retinopathy and is well-recognized as a major proinflammatory mediator of diabetic vascular disease [25]. Our findings in Figure 7 confirm that ropivacaine-mediated suppression of TxNIP/NLRP3 inflammasome activation inhibits expression of IL-1? and IL-18, thereby exerting a significant anti-inflammatory effect in endothelial cells exposed to injury by high glucose.

SIRT1 is a protective factor that has been shown to inhibit the inflammatory response in endothelial cells by suppressing activation of the NLRP3 inflammasome and subsequent release of IL-1?. SIRT1 was also shown to be reduced in endothelial cells in response to lipopolysaccharide and adenosine triphosphate exposure [34]. Concordantly, our findings show that exposure to high glucose reduces expression of SIRT1 and that ropivacaine treatment can ameliorate this effect (Figure 8). Furthermore, the results of our SIRT1 knockdown experiment in Figure 9 show that SIRT1 knockdown abolished the inhibitory effects of SIRT1 on activation of the NLRP3 inflammasome. Importantly, these findings indicate that SIRT1 signaling plays a vital role in the beneficial effects of ropivacaine treatment demonstrated by the other experiments performed in this study. Additional research is required to better elucidate the potential protective effects of ropivacaine against high glucose-induced endothelial dysfunction and inflammation as well as the underlying mechanisms.

References

[1]
Abdul-Ghani M, DeFronzo RA, Del Prato S, Chilton R, Singh R, Ryder RE. Cardiovascular disease and type 2 diabetes: has the dawn of a new era arrived?. Diabetes Care. 2017 Jul 1;40(7):813-20.
[2]
Altabas V. Diabetes, endothelial dysfunction, and vascular repair: what should a diabetologist keep his eye on?. International journal of endocrinology. 2015;2015.
[3]
Domingueti CP, Dusse LM, das Graças Carvalho M, de Sousa LP, Gomes KB, Fernandes AP. Diabetes mellitus: the linkage between oxidative stress, inflammation, hypercoagulability and vascular complications. Journal of Diabetes and its Complications. 2016 May 1;30(4):738-45.
[4]
Chen J, Brodsky SV, Goligorsky DM, Hampel DJ, Li H, Gross SS, Goligorsky MS. Glycated collagen I induces premature senescence-like phenotypic changes in endothelial cells. Circulation research. 2002 Jun 28;90(12):1290-8.
[5]
De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH, Vanhoutte PM. Endothelial dysfunction in diabetes. British journal of pharmacology. 2000 Jul 1;130(5):963-74.
[6]
Gill EK, Edgar KS, Wilson AJ, Patterson E, Grieve DJ. 144 Hyperglycaemia alters NOX4 nadph oxidase-mediated endothelial cell regulation in vitro.
[7]
Wang L, Li X, Zhang Y, Huang Y, Zhang Y, Ma Q. Oxymatrine ameliorates diabetes?induced aortic endothelial dysfunction via the regulation of eNOS and NOX4. Journal of cellular biochemistry. 2018 Nov 19.
[8]
Shah A, Xia L, Goldberg H, Lee KW, Quaggin SE, Fantus IG. Thioredoxin-interacting protein mediates high glucose-induced reactive oxygen species generation by mitochondria and the NADPH oxidase, Nox4, in mesangial cells. Journal of Biological Chemistry. 2013 Mar 8;288(10):6835-48.
[9]
Abderrazak A, El Hadri K, Bosc E, Blondeau B, Slimane MN, Büchele B, Simmet T, Couchie D, Rouis M. Inhibition of the inflammasome NLRP3 by arglabin attenuates inflammation, protects pancreatic ?-cells from apoptosis, and prevents type 2 diabetes mellitus development in ApoE2Ki mice on a chronic high-fat diet. Journal of Pharmacology and Experimental Therapeutics. 2016 Jun 1;357(3):487-94.
[10]
Lebreton F, Berishvili E, Parnaud G, Rouget C, Bosco D, Berney T, Lavallard V. NLRP3 inflammasome is expressed and regulated in human islets. Cell death & disease. 2018 Jun 25;9(7):726.
[11]
Van de Veerdonk FL, Netea MG, Dinarello CA, Joosten LA. Inflammasome activation and IL-1? and IL-18 processing during infection. Trends in immunology. 2011 Mar 1;32(3):110-6.
[12]
Grishman EK, White PC, Savani RC. Toll-like receptors, the NLRP3 inflammasome, and interleukin-1? in the development and progression of type 1 diabetes. Pediatric research. 2012 Jun;71(6):626.
[13]
Wang Y, Han Z, Fan Y, Zhang J, Chen K, Gao L, Zeng H, Cao J, Wang C. MicroRNA-9 inhibits NLRP3 inflammasome activation in human atherosclerosis inflammation cell models through the JAK1/STAT signaling pathway. Cellular Physiology and Biochemistry. 2017;41(4):1555-71.
[14]
Luo B, Li B, Wang W, Liu X, Liu X, Xia Y, Zhang C, Zhang Y, Zhang M, An F. Rosuvastatin alleviates diabetic cardiomyopathy by inhibiting NLRP3 inflammasome and MAPK pathways in a type 2 diabetes rat model. Cardiovascular drugs and therapy. 2014 Feb 1;28(1):33-43.
[15]
Yang SM, Ka SM, Wu HL, Yeh YC, Kuo CH, Hua KF, Shi GY, Hung YJ, Hsiao FC, Yang SS, Shieh YS. Thrombomodulin domain 1 ameliorates diabetic nephropathy in mice via anti-NF-?B/NLRP3 inflammasome-mediated inflammation, enhancement of NRF2 antioxidant activity and inhibition of apoptosis. Diabetologia. 2014 Feb 1;57(2):424-34.
[16]
Becker DE, Reed KL. Local Anesthetics: Review of Pharmacological Considerations. Anesth Prog. 2012; 59(2): 90–102.

[17]
Jayaram P, Kennedy DJ, Yeh P, Dragoo J. Chondrotoxic Effects of Local Anesthetics on Human Knee Articular Cartilage?A Systematic Review. PM&R. 2018 Nov 19.
[18]
Behnaz F, Soltanpoor P, Teymourian H, Tadayon N, Reza G. Sympatholytic and Anti-Inflammatory Effects of Ropivacaine and Bupivacaine After Infraclavicular Block in Arterio Venous Fistula Surgery. Anesth Pain Med. 2019 Jan 15.
[19]
Wu L, Li L, Wang F, Wu X, Zhao X, Xue N. Anti-Inflammatory Effect of Local Anaesthetic Ropivacaine in Lipopolysaccharide-Stimulated RAW264. 7 Macrophages. Pharmacology. 2019;103(4-5):228-35.
[20]
Ota H, Akishita M, Eto M, Iijima K, Kaneki M, Ouchi Y. Sirt1 modulates premature senescence-like phenotype in human endothelial cells. Journal of molecular and cellular cardiology. 2007 Nov 1;43(5):571-9.
[21]
Li Y, Yang X, He Y, Wang W, Zhang J, Zhang W, Jing T, Wang B, Lin R. Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells. Immunobiology. 2017 Mar 1;222(3):552-61.
[22]
Sun X, Jiao X, Ma Y, Liu Y, Zhang L, He Y, Chen Y. Trimethylamine N-oxide induces inflammation and endothelial dysfunction in human umbilical vein endothelial cells via activating ROS-TXNIP-NLRP3 inflammasome. Biochemical and biophysical research communications. 2016 Dec 2;481(1-2):63-70.
[23]
Younis WH, Al-Rawi NH, Mohamed MA, Yaseen NY. Molecular events on tooth socket healing in diabetic rabbits. British Journal of Oral and Maxillofacial Surgery. 2013 Dec 1;51(8):932-6.
[24]
Cosentino F, Lüscher TF. Endothelial dysfunction in diabetes mellitus. Journal of cardiovascular pharmacology. 1998;32:S54-61.
[25]
Liu Y, Costa MB, Gerhardinger C. IL-1? is upregulated in the diabetic retina and retinal vessels: cell-specific effect of high glucose and IL-1? autostimulation. PloS one. 2012 May 16;7(5):e36949.
[26]
Suchanek H, Mysliwska J, Siebert J, Wieckiewicz J, Hak L, Szyndler K, Kartanowicz D. High serum interleukin-18 concentrations in patients with coronary artery disease and type 2 diabetes mellitus. European cytokine network. 2005 Oct 30;16(3):177-85.
[27]
Tang ST, Wang F, Shao M, Wang Y, Zhu HQ. MicroRNA-126 suppresses inflammation in endothelial cells under hyperglycemic condition by targeting HMGB1. Vascular pharmacology. 2017 Jan 1;88:48-55.
[28]
Wohlrab P, Kaun C, Saleh L, Knöfler M, Tretter V, Klein KU. Ropivacaine Causes Inflammation and Apoptosis in Human Umbilical Vein Endothelial Cells and Human Placental Trophoblasts. AINS-Anästhesiologie Intensivmedizin Notfallmedizin Schmerztherapie. 2018 Oct;53(S 01):18.
[29]
Piegeler T, Votta-Velis EG, Bakhshi FR, Mao M, Carnegie G, Bonini MG, Schwartz DE, Borgeat A, Beck-Schimmer B, Minshall RD. Endothelial Barrier Protection by Local AnestheticsRopivacaine and Lidocaine Block Tumor Necrosis Factor-?–induced Endothelial Cell Src Activation. Anesthesiology: The Journal of the American Society of Anesthesiologists. 2014 Jun 1;120(6):1414-28.
[30]
Yu Y, Tang D, Kang R. Oxidative stress-mediated HMGB1 biology. Frontiers in physiology. 2015 Apr 7;6:93.
[31]
Liu R, Luo Q, You W, Jin M. MicroRNA-106 attenuates hyperglycemia-induced vascular endothelial cell dysfunction by targeting HMGB1. Gene. 2018 Nov 30;677:142-8.
[32]
Perrone L, Devi TS, Hosoya KI, Terasaki T, Singh LP. Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions. Journal of cellular physiology. 2009 Oct;221(1):262-72.
[33]
Li Y, Yang J, Chen MH, Wang Q, Qin MJ, Zhang T, Chen XQ, Liu BL, Wen XD. Ilexgenin A inhibits endoplasmic reticulum stress and ameliorates endothelial dysfunction via suppression of TXNIP/NLRP3 inflammasome activation in an AMPK dependent manner. Pharmacological research. 2015 Sep 1;99:101-15.
[34]
Li Y, Yang X, He Y, Wang W, Zhang J, Zhang W, Jing T, Wang B, Lin R. Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells. Immunobiology. 2017 Mar 1;222(3):552-61.
Legends
Figure 1. Ropivacaine ameliorated high glucose- induced production of mitochondrial ROS in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with high glucose (25 mM) in the presence or absence of 0.25% and 0.5% ropivacaine for 24 h. Mitochondrial ROS (1, 3.4, 2.1, 1.5) was measured with MitoSOX (*, #, $, P

Did you like this example?

3219

205