Major findings from the present study demonstrate that, NADPH/NOS-dependent NO generation was crucial for damaged angiogenesis of RAECs triggered by high glucose. Since L-arginine known as a NO donor, vitamin C acting as an antioxidant, we developed a new strategy in which L-arginine combined with vitamin C accelerates the deficiency of angiogenesis via mediating NADPH/NOS/NO/VEGF signaling pathway. Simultaneously, we elucidated that L-arginine and vitamin C co-administration elicited proangiogenic role after AMI injury under T2DM condition.
Angiogenesis, known as new blood vessel formation from existing vessels, supplies impaired tissue adequate blood flow and oxygenation. The higher rate of myocardial angiogenesis is a compensatory mechanism secondary to the ischemia developed by coronary artery disease and other myocadiac diseases. It has been increasingly noticed that therapeutic angiogenesis is an attractive tool for diabetic cardiac ischemia by improving blood flow and providing oxygen to form new coronary collateral vessels. Previous studies revealed that diabetics exhibit a diminished angiogenic response to myocardial ischemia likely as a result of hyperglycemic environment. The process of neovascularization deficiency of myocardial infarction caused by hyperglycemia is demonstrated by Fig. 1. Previous evidence has revealed that the single administration of L-arginine or Vitamin C exquisitely enhances vascularity in ischemic hearts, acting as a protective role on cardiovascular diseases, respectively. Thus, we hypothesized that the combination therapy of L-arginine and vitamin C is a promising therapeutic option that plays a better myocardial protection effect than single use hyperglycemia-impaired angiogenic response in diabetic AMI. The proliferation and migration of endothelial cells (ECs) is the initial process of coronary collateral vessel formation, thus we extracted primary rat aorta endothelial cells (RAECs) to implement in vitro experiments. Long been regarded as the root cause of on cardiovascular complications under diabetes mellitus (DM), exposure to hyperglycemia leads to angiogenic inadequacy and other endothelial dysfunctions in DM. First, we addressed whether high glucose stimulation distributes angiogenesis in RAECs in vitro and the role of L-arginine and vitamin C co-treatment on it after high glucose incubation. Following high glucose stimulation for 48 h, we treated RAECs with media containing combination of L-arginine with vitamin C or single drug for 12 h. By detecting cell viability assay to determine proliferation, wound scratch assay to determine EC migration and in vitro sprouting assay to determine tube formation ability, it was demonstrated that conjunctive treatment of L-arginine with vitamin C promoted EC proliferation and migration, accelerated insufficient angiogenesis disrupted by high glucose incubation. As the most pivotal angiogenic factor, VEGF is indispensable for driving endothelial cells proliferation, migration and sprouting to enhance new blood vessels formation. After treated with L-arginine and vitamin C, the expression of VEGF was markedly upregulated compared to reduction elicited by high glucose, indicating that L-arginine with vitamin C co-administration was potential in normalizing VEGF-derived angiogenesis and capillary formation impaired by high glucose. Notably, the effect of incubation with L-arginine or vitamin C alone on EC angiogenesis was not so obvious as that in united application.
The mechanism of L-arginine with vitamin C co-treatment in protecting against hypo-angiogenesis injury in T2DM is summarized in Fig. 2. It is well-acknowledged that NO plays an integral role of endothelial homeostasis, mediating the downstream VEGF activity in angiogenesis[19, 29]. As a critical mediator of angiogenesis, NO is mostly produced by NO synthase (NOS) isoforms eNOS and iNOS in cardiovascular system. It has been documented that eNOS plays a pivotal role in NO bioavailability, as it provides primary endogenous amount of NO, whereas iNOS acts mainly as a cytotoxic induced by oxidative stress and inflammatory conditions. Previous researches[21, 30, 38] have proved that PI3K/AKT/eNOS signaling pathway actively regulates VEGF-induced neovascularization. Since L-arginine is a substrate for eNOS that produces NO and vitamin C acts as a partially mediator of NO activation, we continued to investigate whether NOS/NO signaling was the potential molecular mechanisms of VEGF-induced angiogenic regulation of L-arginine with vitamin C co-administration. In this study, by detecting supernatant NO level in RAECs, phosphorylated-eNOS, total eNOS, iNOS protein expression and supernatant content, we demonstrated that combination treatment of L-arginine with vitamin C indeed stimulated NO production likely attributed to eNOS phosphorylation motivation and iNOS inhibition. Additionally, abolishment of eNOS, iNOS and total NOS pointed towards a fundamental role of NOS/NO within angiogenetic process. Taken together, these observations suggest that the hyperglycemia-induced angiogenesis deficiency is in part due to a derangement of the NOS/NO pathway, whereas co-treatment of L-arginine and vitamin C could play a pro-angiogenic role by simultaneously activating eNOS and inhibiting iNOS.
It is well-accepted that the presence of hyperglycemia in diabetes is associated with oxidative stress stimulation, aggravates myocardial ischemia injury. It has been reported that by eliminating O2-, oxidative stress stimulated ROS and RNS accumulation largely resulted from DM, and in turn, interrupts normal metabolism and slows down angiogenesis speed in myocardium. Meanwhile, it was also demonstrated that NOS activity is implicated with over-produced O2-, namely eNOS inactivation and iNOS activation.
A family of NADPH oxidases is regarded as the predominant source of O2-, which disturbs angiogenesis process in both cultured cells and in vivo models of neovascularization. Given the potential therapeutic mechanism of combination therapy, we speculated that though L-arginine appears to be beneficial to reducing oxidative stress on vascular endothelial function in cardiovascular diseases, this effect is modest and hard to work especially at physiological states.
Vitamin C, an antioxidant that scavenges ROS and depletes the excessive accumulation of O2-, may restore NOS/NO pathway activity to promote angiogenesis. This possibility seems to be confirmed by the present study by observing oxidative stress level in vitro. In addition, we verified that NADPH-derived O2- generation is probably the main mechanism responsible for inactivating downstream NO-derived angiogenesis in diabetes. In the context of L-arginine plus vitamin C, this study supports the concept that the amplification of angiogenesis in this combination treatment is associated with the fact that, L-arginine mainly plays the pro-angiogenic role by ameliorating the expression of eNOS, iNOS and NO, ultimately enhancing collateral growth. However, this effect can be partially reduced resulting from oxidative stress. In the capacity of vitamin C as an antioxidant, it lowered the amount of NADPH-derived O2- generation to preserve activation of angiogenic eNOS/NO signaling pathway, amplifying the beneficial effect of L-arginine.
It has been understood that when suffering external damage like hyperglycemia, O2- production can rapidly react with NO, resulting in the generation of peroxynitrite (ONOO-)[8, 34], under myocardial injury. The production of ONOO- increased by DM-induce NADPH is an intense cytotoxic oxidant that in turn, deteriorates stress states and aggravates downstream angiogenesis in diabetic myocardial injury. Alternatively, ONOO- increases further predisposes the heart to NOS uncoupling, which is associated with eNOS inactivation. According to previous findings, excessive exogenous L-arginine supplement may shift to an injurious scenario with increased production of ONOO-. Nevertheless, ONOO- generation can be abrogated by vitamin C due to the effect on O2- scavenging. Similar conclusions have been reached in the present study, in which co-supplementation of L-arginine and vitamin C lessened the level of ONOO- and increased NO bioavailability. This notion explains the phenomenon that even treated with vitamin C alone, it mildly activated eNOS activity. Therefore, another advantage of the combination application of L-arginine with vitamin C is, oral administration of vitamin C fills the gap of L-arginine that produces ONOO- to decrease adverse reactions.
It may be worth noting that apart from the pharmacodynamics interactions, the mutual effects on pharmacokinetics are deserved further assessment. We focus our interest on the status of intra-cellular arginine metabolism in affecting endothelial angiogenesis, more specifically, governing eNOS activity and NO production. It has been reported that as the predominant contributor among different arginine transporters in endothelial cells, cationic amino acid transporter 1 (CAT-1) can be modulated by multiply redox-sensitive mechanisms. Interestingly, Schwartz et al has elaborated the underlying interaction of CAT-1 with eNOS activity that CAT-1 specially delivers transported arginine to membrane-bound eNOS. Thus, in our study, we detected the effect of high glucose stimulation combination treatment of L-arginine with vitamin C on arginine uptake by measuring CAT-1 expression in RAECs. The result suggested that when combined with vitamin C, exogenous L-arginine was transported into cells more active compared with L-arginine single treatment, suggesting that vitamin C improved the availability and utilization efficiency of L-arginine. Nevertheless, incubation with vitamin C alone showed limited benefits possibly attributed to the extra-cellular L-arginine concentrations. To this end, this described pharmaceutical interactions may pave the way for the potential amplified therapeutic effect of this promising combined therapy.
In addition, the therapeutic effects and mechanisms of L-arginine plus vitamin C must be examined in vivo experiments. In our study, AMI rats under the condition of T2DM impaired collateral formation in myocardium, providing a well-recognized model of angiogenesis deficiency. By the changes in biochemical criterions, we found that L-arginine with vitamin C co-administration inhibited the level of blood lipid and decreased the blood glucose in T2DM injury. The exogenous administration of L-arginine with vitamin C improved cardiac function by the detection of LVEF and LVFS, along with the result of LDH serum level. Nevertheless, the application of L-arginine or vitamin C alone has not induced significant effect of improved cardiac function, which is in agreement with results in vitro. What’s more, the relaxation of aortic rings responding to endothelium-dependent vasodilator such as acetylcholine but not to an endothelium-independent vasodilator such as SNP was impaired in T2DM rats as well as T2DM + AMI rats, whereas the impairment could be attenuate by L-arginine and vitamin C co-intervention. As impaired vasoconstrictor responses to phenylephrine were restored by treatment of aortic rings with L-arginine and L-NNA, this implies that basal and inducible NO production is enhanced in L-arginine plus vitamin C group. In line with the in vitro ratiocinations, by observing the results of MVD assay, VEGF immunohistochemistry and TTC staining, we concluded that L-arginine + vitamin C co-supplementation reduced infarction size, promoted angiogenesis and arteriogenesis in DM-induced impairment of coronary collateral formation, showing an amplify effects compared with single treatment. At the same time, the results from plasma NO levels, eNOS and iNOS immunohistochemistry as well as their serum contents, can further prove findings in vitro that a functional association between L-arginine and vitamin C combined therapy and NOS/NO-induced angiogenesis. Our in vivo results also supported the in vitro data showing that L-arginine and vitamin C co-treatment decreased MDA serum level and reduced NADPH expression, confirming the therapeutic mechanism of NADPH-derived oxidative stress inhibition.
Endothelial cell angiogenesis contributes to endothelial function impairment resulting in vascular dysfunction T2DM. Thus, to further verify the role of L-arginine and vitamin C in response to vascular dysfunction induced by diabetic AMI, vasodilation test was assayed in rats. As endothelial function is determined by Ach-induced relaxation, we monitored Ach-induced endothelium-dependent vasodilatation. As indicated in Fig. 9a-c, hyperglycemia impaired endothelium-dependent relaxation obviously in T2DM and T2DM + AMI group. After 2 weeks treatment of L-arginine and vitamin C, the reduction of Ach-induced vasodilatation was been reversed. According to the analytical data, combination therapy enhanced the endothelium-dependent relaxation more significantly in T2DM rats than that treated L-arginine or vitamin C alone. Additionally, vascular response to SNP, a NO donor, was tested, which evoked endothelium-independent vasodilatation in thoracic aorta. By measuring endothelium-independent relaxation, it can be inferred that the normal function of vascular smooth muscle cells didn’t been damaged in terms of NO.
As NO plays the vital role of endothelial function, NO release under basal and inducible conditions were also detected. The concentration-response curves to phenylephrine on the aortic rings among 6 groups were showed in Fig. 9d-g. Pretreatment with L-Arginine, the NO synthesis substrate, weakened the concentration-response to phenylephrine in the endothelium-intact aortic rings. On the contrary, after the incubation with the NOS inhibitor L-NNA, the contractile response to phenylephrine was strengthened. As illustrated in, T2DM as well as T2DM + AMI rats showed reduced ΔAUC. Under the influence of L-arginine and vitamin C co-treatment, the decreased ΔAUC was reversed, implying that combination treatment availably restored hyperglycemia-impaired NO basal release and induced release. Taken together, we testified that co-application of L-arginine and vitamin C ameliorated cardiac trauma together with vascular dysfunction of diabetic AMI rats.