The Emerging Roles of Chromogranins and Derived Polypeptides in Atherosclerosis, Diabetes, and Coronary Heart Disease

Chromogranin A (CgA), B (CgB), and C (CgC), the family members of the granin glycoproteins, are associated with diabetes. These proteins are abundantly expressed in neurons, endocrine, and neuroendocrine cells. They are also present in other areas of the body. Patients with diabetic retinopathy have higher levels of CgA, CgB, and CgC in the vitreous humor. In addition, type 1 diabetic patients have high CgA and low CgB levels in the circulating blood. Plasma CgA levels are increased in patients with hypertension, coronary heart disease, and heart failure. CgA is the precursor to several functional peptides, including catestatin, vasostatin-1, vasostatin-2, pancreastatin, chromofungin, and many others. Catestatin, vasostain-1, and vasostatin-2 suppress the expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in human vascular endothelial cells. Catestatin and vasostatin-1 suppress oxidized low-density lipoprotein-induced foam cell formation in human macrophages. Catestatin and vasostatin-2, but not vasostatin-1, suppress the proliferation and these three peptides suppress the migration in human vascular smooth muscles. Chronic infusion of catestatin, vasostatin-1, or vasostatin-2 suppresses the development of atherosclerosis of the aorta in apolipoprotein E-deficient mice. Catestatin, vasostatin-1, vasostatin-2, and chromofungin protect ischemia/reperfusion-induced myocardial dysfunction in rats. Since pancreastatin inhibits insulin secretion from pancreatic β-cells, and regulates glucose metabolism in liver and adipose tissues, pancreastatin inhibitor peptide-8 (PSTi8) improves insulin resistance and glucose homeostasis. Catestatin stimulates therapeutic angiogenesis in the mouse hind limb ischemia model. Gene therapy with secretoneurin, a CgC-derived peptide, stimulates postischemic neovascularization in apolipoprotein E-deficient mice and streptozotocin-induced diabetic mice, and improves diabetic neuropathy in db/db mice. Therefore, CgA is a biomarker for atherosclerosis, diabetes, hypertension, and coronary heart disease. CgA- and CgC--derived polypeptides provide the therapeutic target for atherosclerosis and ischemia-induced tissue damages. PSTi8 is useful in the treatment of diabetes.


Introduction
Coronary heart disease is now the leading cause of death worldwide [1]. The risk factors for coronary heart disease involve hypercholesteremia, diabetes, hypertension, obesity, and metabolic syndrome [1]. Coronary heart disease exhibits myocardial ischemia and dysfunction induced by significant stenosis in coronary arteries that supply the heart with blood [1]. It is usually caused by atherosclerosis, which is a chronic inflammatory disease with a buildup of cholesterol-rich plaques inside the artery walls [1]. Atherosclerosis is characterized by a complex multicellular process [2], and is triggered by arterial injury-induced endothelial inflammation. This results in the formation of intimal atheroma and plaque caused by oxidized low-density lipoprotein (LDL)-induced macrophage foam

Cgs-Derived Polypeptides
CgA was identified as an acidic protein costored and coreleased with ATP and catecholamines in chromaffin granules of neuroendocrine cells in the adrenal medulla [25]. CgA is also present in other secretory vesicles of neuronal and endocrine tissues including the pancreatic islet, in addition to keratinocytes, cardiomyocytes, ECs, and macrophages [3,19,20].

Biomarker for Diabetes, Metabolic Syndrome, and Cardiovascular Disease
The  CgB is abundantly expressed in many neurons and endocrine cells [3]. After synthesis, CgB is posttranslationally O-glycosylated and stored to large secretory vesicles [3]. Within granules, CgB is proteolytically processed at diabasic Lys-Arg and monobasic Arg sites to several proteins of intermediate size and small peptides [3]. From bovine CgB (646 amino acids), the 13-amino acid peptide secretolytin (CgB614-626) was identified, and has the biological activity as an antibacterial agent [39]. Secretolytin has been also found in human blood [40].
The roles of Cgs and their cleavage products as the biomarkers and pathogenesis of diabetes and atherosclerotic cardiovascular diseases are described in the following chapters.

Biomarker for Diabetes, Metabolic Syndrome, and Cardiovascular Disease
The Data are shown as mean ± SD. NE = not examined.
Since CgA is even more stable compared with cathecholamines in the circulating blood, its plasma levels reflect the sympathetic tone and adrenomedullary system activity, which are altered in coronary artery disease (CAD), heart failure, and hypertension [37]. Circulating levels of CgA are increased and associated with the mortality of patients with CAD [52][53][54][55] (Table 2). In CAD patients, plasma CgA levels rise even higher in the presence of heart failure [55]. Plasma levels of vasostatin-1 are positively associated with carotid atherosclerosis [56]. In contrast, circulating levels of vasostatin-2 and catestatin are significantly decreased in patients with CAD compared with healthy control groups of patients [19,50,57,58] (Table 2). Serum levels of vasostatin-2 are also decreased in patients with ischemic chronic heart failure [59]. However, catestatin levels are increased at the onset of acute myocardial infarction, which is correlated with norepinephrine levels [60], and leads to adverse events [61]. In addition, the increase in catestatin levels also contributes to coronary collateral development and left ventricular remodeling [62][63][64]. Plasma levels of vasostatin-1 and secretolytin are increased in patients with coronary artery bypass graft surgery [40]. Plasma levels of CgA, catestatin, and pancreastatin are significantly increased in patients with hypertension compared with healthy control subjects [65][66][67] (Table 2). There are higher plasma levels of CgA, CgB, catestatin, VIF, and secretoneurin in patients with heart failure compared with healthy control groups of patients [23,42,[68][69][70]. Serum levels of CgA and CgB are significantly higher in the presence of carcinoid heart disease among patients with neuroendocrine tumors [71]. Levels of plasma CgA are much higher in patients with dilated cardiomyopathy or hypertrophic cardiomyopathy than the levels in the healthy controls [72]. Plasma vasostatin-1 levels are increased in patients with Takayasu arteritis [73]. High levels of secretoneurin are associated with the increased risk of mortality in patients with heart failure, aortic stenosis, or those patients undergoing various cardiac surgeries [74][75][76].
CgA is detected at higher levels in the saliva of type 2 diabetic patients compared with healthy and nondiabetic subjects [77,78]. In patients with type 2 diabetes, the high levels of salivary CgA are associated with periodontal damage [78]. Therefore, CgA in saliva may be a biomarker for oral health in patients with type 2 diabetes. The levels of CgA, CgB, and CgC in the vitreous humor are higher in patients with diabetic retinopathy compared with nondiabetic subjects [79].
As clinical biomarkers, CgA, CgB, CgC, and derived polypeptides are closely associated with atherosclerotic cardiovascular diseases and diabetes. Next, this review describes their cardiovascular effects as well as the molecular and cellular mechanisms of their antiatherosclerotic and anti-diabetic effects, and expands to their emerging roles in therapeutic strategies against atherosclerotic cardiovascular diseases and diabetes.
In addition to cardiovascular protective effects, the atheroprotective effects of Cgsderived polypeptides in vitro and in vivo are especially described in the next Chapter.

Atherosclerosis
Atherosclerosis is triggered by arterial injury-induced inflammation. This process includes hyperpermeability, proliferation of ECs followed by the formation of atheroma-tous plaques involving oxidized LDL-induced foam cell formation in monocyte-derived macrophages, migration and proliferation of VSMCs, and extracellular matrix production by VSMCs [2] (Figure 2). As described above, in the formation and development of atherosclerosis in the arterial walls, three types of vascular cells, such as ECs, macrophage, and VSMCs, are known as the major players. Therefore, the effects of CgA-and CgCderived polypeptides on these vascular cells are described in detail in the following sections. cardiac dysfunction and inhibits cardiac remodeling following myocardial infarction [93].
In addition to cardiovascular protective effects, the atheroprotective effects of Cgsderived polypeptides in vitro and in vivo are especially described in the next Chapter.

Atherosclerosis
Atherosclerosis is triggered by arterial injury-induced inflammation. This process includes hyperpermeability, proliferation of ECs followed by the formation of atheromatous plaques involving oxidized LDL-induced foam cell formation in monocyte-derived macrophages, migration and proliferation of VSMCs, and extracellular matrix production by VSMCs [2] (Figure 2). As described above, in the formation and development of atherosclerosis in the arterial walls, three types of vascular cells, such as ECs, macrophage, and VSMCs, are known as the major players. Therefore, the effects of CgA-and CgC-derived polypeptides on these vascular cells are described in detail in the following sections.

ECs
Early atherosclerosis features vascular injury-induced changes in endothelial structure and barrier function that affect the traffic of molecules and solutes between the vessel lumen and the vascular wall [2]. Proatherogenic stimuli and cardiovascular risk factors, such as hypertension, dyslipidemia, diabetes, and smoking, increase endothelial permeability [94]. These factors share a common signaling denominator: an imbalance in the production/disposal of reactive oxygen species (ROS), broadly termed oxidative stress [94]. As a consequence of the activation of enzymatic systems leading to ROS overproduction, proatherogenic factors lead to a proinflammatory status that translates to changes in gene expression and functional rearrangements, including changes in the transendothelial transport of LDL [94]. Oxidation of LDL by ROS triggers the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in ECs [2]. Circulating monocytes attach to ECs and subsequently infiltrate into the intima [2]. In addition, EC proliferation contributes to the formation of intimal lesions [95]. The migration and proliferation of ECs are important phenomena for angiogenesis and also atherogenesis.

Macrophages
Monocytes migrate into the subendothelial space, and then differentiate to macrophages [2]. Macrophages phagocytose oxidized LDL and transform into foam cells [2]. Foam cell formation depends on the homeostatic balance between the uptake of oxidized LDL via CD36, the efflux of free cholesterol controlled by the ATP-binding cassette transporter A1 (ABCA1), and cholesterol esterification by acyl coenzyme A: cholesterol acyltransferase-1 (ACAT-1) [106].
Catestatin and secretoneurin stimulate the migration of human monocytes [107,108]. These findings suggest that the two peptides contribute to the biodefence and inflammatory response in vascular walls. Catestatin and vasostatin-1 induce the anti-inflammatory phenotype and suppress the inflammation in human macrophages [19,20,109].

VSMCs
VSMCs contribute to the progression of atherosclerotic plaque through their migration, proliferation, and the production of ECM components, such as collagens, matrix metalloproteinases, fibronectin, and elastin. In particular, collagens promote the formation of the fibrous cap of atherosclerotic plaques [110]. The fibrous cap contributes to stabilizing atherosclerotic plaque to prevent its rupture. Elastin plays an essential role in the maintenance of vascular elasticity [111].

Murine Models of Atherosclerosis
The in vivo effects of CgA and its derived peptides on atherosclerosis have been evaluated in murine models with their exogenous infusion and endogenous deficiency. A chronic infusion of catestatin, vasostatin-1, or vasostatin-2 suppresses the development of atherosclerosis of the aorta in apolipoprotein E-deficient mice [19][20][21]. These anti-atherosclerotic effects are attributed to the molecular and cellular protective effects against atherosclerosis, as described above. Catestatin also attenuates insulin resistance, hypertension, and obesity in murine models, and contributes to the prevention of metabolic syndrome [116]. CgA-knockout mice reveal hypertension, high plasma catecholamine and adiponectin levels, and lower interleukin-6 and lipid levels compared with wild type mice [117]. CgA-knockout mice also exhibit enhanced insulin sensitivity despite obesity [118]. These findings suggest that CgA prevents the development of atherosclerosis. Next, the preventive effects of CgA and derived peptides on atherosclerotic cardiovascular diseases in murine models are described.
In addition, gene therapy with the CgC-derived peptide secretoneurin ameliorates hind limb and myocardial ischemia without influencing systemic atherosclerosis in apolipoprotein E-deficient mice [124]. Secretoneurin protects skeletal muscle and myocardium against ischemic injury and apoptosis [42]. Secretoneurin gene therapy has a variety of effects. It stimulates coronary angiogenesis, improves left ventricular function, and inhibits myocardial remodeling in a rat model of myocardial infraction [93]. Oral administration of secretoneurin enveloped in nanoparticles restores blood flow in the mouse hind limb ischemia model [125]. Secretoneurin gene therapy also stimulates postischemic neovascularization in streptozotocin-induced diabetic mice [126], and improves diabetic neuropathy in db/db mice [127]. Secretoneurin suppresses hypertrophy and oxidative stress via AMPactivated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK) pathways in mouse cardiomyocytes [128]. Secretoneurin promotes neuroprotection and neuroplasticity via the Janus kinase-2/signal transducer and activator of transcription-3 pathway in murine models of stroke [129,130].

Diabetes
CgA is known to play a significant role in the pathogenesis and development of type 1 diabetes [10]. In vivo and in vitro experiments have determined that the function of CgB relates to the physiological secretion of insulin. CgB regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet β-cells [34]. Catestatin suppresses hepatic glucose production and improves insulin sensitivity [131]. WE-14 (the abbreviation comes from N-and C-terminal amino acids and the length of the molecule) and human CgA10-19 serve as an autoantigen for both CD4+ and CD8+ β-cell-destructive diabetogenic T-cell clones in type 1 diabetes [132,133]. A recent study has identified a CgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of CgA, as the BDC2.5 TCR epitope [134]. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T-cells and induces an interferon-γ response [134]. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with CgA29-42 peptide transferred diabetes into NOD/SCID mice [134].
Pancreastatin inhibits insulin secretion from pancreatic islet β-cells [12] and also regulates glucose, lipid, and protein metabolism in liver and adipose tissues [135]. Pancreastatin inhibits glucose uptake and glycogen synthesis but stimulates gluconeogenesis in hepatocytes [135]. Pancreastatin inhibits glucose uptake and glycogen synthesis in adipocytes [136]. Pancreastatin increases lipid droplets and ROS production in 3T3-L1 adipocyte cells [137]. These effects of pancreastatin are exerted via phosphatidylinositol 3-kinase/protein kinase C and glycogen synthase kinase-3 [136]. Pancreastatin plays a significant role in obesity-induced insulin resistance [138]. In healthy humans, a standard meal increases serum pancreastatin levels [139], and human pancreastatin infusion decreases forearm glucose uptake [140]. An intravenous infusion of human pancreastatin-16 suppresses the elevation of serum insulin levels without glucose overshoot on an oral glucose tolerance test in healthy humans [141]. Pancreastatin may induce the impaired insulin secretion and insulin resistance in the setting of diabetes and/or obesity.
Pancreastatin inhibitor peptide-8 (PSTi8), which consists of 21 amino acids (PEGKGEQEHSQQKEEEEEMAV-amide), exerts antidiabetic effects. These effects have been demonstrated by cell and animal studies [13,[142][143][144]. PSTi8 decreases pancreastatininduced insulin resistance in HepG2 cells (human liver cancer cells) and 3T3-L1 cells (mouse adipocyte cells) [13,142]. PSTi8 increases glucose uptake via enhanced glucose transporter type 4 in L6 cells (rat skeletal myoblast cells) [13,143] and decreases hepatic glucose release [144]. The treatment with PSTi8 increases insulin sensitivity in db/db, high fat and fructose-fed streptozotocin-induced insulin resistance mice [13]. PSTi8 improves the obesity-associated insulin resistance and inflammation in skeletal muscle [143], and improves hyperinsulinemia-induced obesity and inflammation-mediated insulin resistance in adipose tissue via inhibition of ERK/c-Jun N-terminal protein kinase pathways [137]. PSTi8 also improves dexamethasone-induced fatty liver by suppressing lipid deposition and oxidative stress through the glucose-regulated protein-78 followed by the AMPK pathway [144]. Further clinical studies are needed to clarify the efficacy of PSTi8 in the treatment of patients with diabetes and obesity.

Conclusions
CgA and derived polypeptides are the convincing biomarkers for atherosclerosis, diabetes, hypertension, and cardiovascular diseases. Circulating levels of CgA and pancreastain are high in type 1 and type 2 diabetes, respectively, because CgA is one of pathogeneses of type 1 diabetes, and pancreastatin induces insulin hyposecretion and insulin resistance. Circulating CgA levels are high in hypertension, CAD, and heart failure that show increments in the sympathetic tone and adrenomedullary system activity. Circu-lating levels of catestatin and vasostatin-2 are low in CAD. As catestatin and vasostatin-2 have atheroprotective effects, their decreased levels may be a risk factor for CAD.
PSTi8 is useful in the treatment of diabetes and metabolic syndrome. Catestatin, vasostatin-1, and vasostatin-2 serve the therapeutic target for atherosclerosis and coronary heart disease. Vasostatin-1 and secretoneurin stimulate ischemia-induced angiogenesis. Catestatin, vasostatin-1, and chromofungin protect ischemic myocardial damage. Cgs and derived polypeptides are a vision of new therapeutic strategies for atherosclerotic and ischemic cardiovascular diseases.