Molecular interaction between CRP-LOX 1 complex and Ligands
Figure 1(a) & (b) illustrates the 3D structure of CRP and LOX 1. The active sites of CRP and LOX 1 were predicted using CASTp as shown in figures 1 (c) & (d). The protein-protein interaction of CRP-LOX1 was carried out using patch dock server and that complex was uploaded in molecular docking server tools. From the results, the CRP-LOX-1 complex was disrupted by the loss of binding site for LOX-1, since OPC monomers occupies that binding onto CRP. Thereby, from the results it was suggestive that OPC could alter the structural confirmation of CRP at the site Ca2+ ions per subunit. We performed this protein -protein-ligand interaction for the first time to illustrate the inhibitory effects of OPC against CRP-LOX1 complex (Figure (e, f & g). The molecular docking study was carried out using lead compounds from C. procera with CRP revealed their anti-analgesic and anti-inflammatory effects . Previous study showed the molecular interactions between strongest interactions of statins with CRP as compared to the standard ligand phosphocholine . Another recent study of Shakour and his group of researchers have revealed the possible molecular interactions between polyphenol curcumin and its derivatives with CRP exhibits the plausible anti-inflammatory and anti-oxidative against diseases . Therefore, OPC could readily inhibit the CRP-LOX1 complex and improves the biological activities. Based on this in silico results, this study extended to in vitro methods to prove the anti-inflammatory properties of OPC.
In vitro studies
The non-toxic concentrations of OPC, OxLDL and CRP on coculture model of ECV 304 cells with THP-1 cells were determined by MTT assay. This cell viability assessment was done in three different environments to fix non-toxic concentrations of OPC, OxLDL and CRP for 24h and 48h. The figure 2(a) illustrates the MTT assay for coculture model of ECV 304 cells with THP-1 cells induced with varying concentrations of CRP (5-25 µg/ml) showed >60% of viability till 48h. Subsequently, the cytotoxicity of OxLDL (figure 2(b)) on coculture model of ECV 304 cells with THP-1 cells was assessed with different concentrations of OxLDL ranging for 5-25 µg/ml of protein. The nontoxic effects of OPC on coculture of ECV 304 cells with THP-1 cells were carried out with various concentrations of OPC (10-200µg) as illustrated in Figure 2(c). From the result, it was observed that increasing concentration of OxLDL resulted in the loss of cell viability. Hence, we fixed 15µg/ml of CRP, 10µg/ml of OxLDL and OPC (100µg/ml) concentration were fixed as a nontoxic concentrations to study the effects of OxLDL mediated endothelial dysfunction and its associated inflammatory signaling pathways. This concentration was used for further study to evaluate the anti-angiogenic, anti-migratory, anti-invasive and anti-apoptotic properties of OPC against CRP and OXLDL induced endothelial dysfunction.
As discussed earlier, active myeloperoxidase (MPO) is released into the vessel as a result of transendothelial migration and involved in endothelial injury, and, internalization of MPO causes oxidative damage and impairs endothelial signaling . Thus, release of MPO in the serum is suggestive of its pro-inflammatory effects on vasoregulatory system . The activity of MPO analysed in the control and experimental groups is represented in Figure 3(a), which showed increased MPO activity in the OxLDL treated group when compared to control. This functional MPO is responsible for the increased transendothelial migration, proinflammatory response in the coculture induced with CRP+OxLDL. Thus, OPC could effectively inhibit MPO activity. Binding of OPC with the OxLDL+ CRP reduces the production of pro-inflammatory mediators.
To further confirm that OPC could efficiently inhibit the trans-endothelial migration, the mRNA expression of trans-endothelial migratory markers was analyzed. L-selectin expressed by the leukocytes plays a pivotal role in the generation of rapid and efficient immune responses during the interaction of circulating leukocytes with the vascular endothelium . L-selectin has been considered as both positive and negative regulator of leukocyte activation and leukocyte recruitment . There are two important and measurable properties of L-selectin; its rapid proteolysis or shedding upon cell activation, and its transition from being monomeric in the plasma membrane to being clustered, following ligand binding, which is a hallmark of downstream signalling . L-selectin is specifically expressed in the monocytes and facilitates the fast rolling of leukocytes on endothelium. The expression of L-selectin was evaluated to assess the adhesion of monocytes on the surface of endothelial cells . Figure 3(b), represents the L-selectin mRNA of OxLDL treated group which showed downregulation, when compared to control group. This could be because of the monocytes stimulated by OxLDL do not undergo rolling, instead tether and adhere onto the endothelial surface and eventually differentiate into macrophages. Loss of L-selectin due to shedding increases leukocyte adhesion and transmigration by increasing leukocyte exposure to the inflamed endothelium and decreasing jerkiness to promote leukocyte activation by outside-in signaling. In the OPC treated group, monocytes were seen floating in the medium and the adhesion was partially attenuated by OPC treatment.
ROS generation is considered as a major atherogenic factor , Nitric oxide (NO) regulates vascular tone and local blood flow, platelet aggregation and adhesion, and leukocyte-endothelial cell interactions. Loss of NO production by the vascular endothelium results in endothelial dysfunction. These results (Figure 4(a)) indicate that loss of NO in the endothelium upon OxLDL assault decreases the eNOS expression. Decreased mRNA expression of eNOS is indicative of the downregulation of Akt in the OxLDL treated group, because eNOS is activated by Akt. The inactive eNOS could enhance the shear stress via loss of NO bioavailability . Hence this study sought to investigate the OxLDL mediated ROS generation and its inhibition by OPC. The antioxidant effect of OPC was evaluated by the formation of MDA- an index for lipid peroxidation and intracellular ROS generation was assessed using oxidation sensitive fluorescent probes, 2′7′-dichlorofluorescin diacetate (DCFH-DA). Recent studies have demonstrated that the increased lipid peroxidation correlates with the increased expression P-Selectin, IL-6, MCP-1 and LOX-1, and involved in the apoptosis of endothelial cells . Figure 4(b) displays the DCF-DA stained endothelial cells and monocytes, which showed increase in fluorescence upon CRP+ OxLDL induction. This result directly points out that OxLDL induces ROS, RNS and H2O2 generation. Thus, the increased ROS and RNS generation mediates the increased transendothelial migration by upregulation of LOX-1, MCP-1, IL-6, IL-1β, L- selectin and TF which, in turn, activates the proinflammatory NFkB, angiogenesis and shear stress. OPC treated group showed decreased fluorescence compared to OxLDL treated group. OPC has strong antioxidative capacity, high affinity for the lipid bilayers of the cell membrane and hence, can easily enter the nuclei of cells. OPC is readily water soluble and oxidizable. Its catechol structure also makes OPC a strong chelator of metal ions. Ellagic acid, anthocyanins and punicalagins also elucidate the attenuation of ROS and thus, prevent endothelial dysfunction and atherosclerosis [40,41].
The formation of capillary tube in in vitro is a significance of endothelial cell differentiation. As discussed earlier, OxLDL and CRP known to direct the loss of angiogenic properties of endothelial via activating apoptotic pathway. The ability of OxLDL to stimulate capillary tube formation in the experimental group was established in this study. As shown in Figure 5(a) & (b), CRP+OxLDL induction led to the formation of capillary-like structures. Normal endothelial cells grow and proliferate and tend to form tubule- like structures. Endothelial cells uptake OxLDL by a well-known scavenger receptor, LOX-1. Hence, the formation of capillary tubes may be dependent on the upregulation of LOX-1 dependent mechanism . The other factors for the formation of capillary-like structure are shear stress, relevant to migration and angiogenesis stimulation; and αvβ3-integrin which plays a critically important role in angiogenesis stimulated by OxLDL . The endothelial cells treated with OPC, showed a significant decrease in the tube forming capacity. Blueberry also inhibited angiogenesis in ECV304 cells by suppressing migration and tube formation . Upon co treatment with OPC under the stimulation of OxLDL and CRP resulted in the formation of capillary tubes when compared to control group of cells significantly.
The wound-healing assay is used to estimate the migration potential of endothelial cells in monolayer culture stimulated by OxLDL. As shown in Figure 6(a&b), group I (control) cells showed increased migration, group II showed significantly reduced cell migration even after 72h, which may be due to the cytotoxic effect of CRP+OxLDL that promotes apoptosis of the endothelial cells. The OPC treated coculture model showed a dramatic increase in the migration of cells from 0 h to 72 h, thus, treatment with OPC could amend the toxic effects of OxLDL and thereby promote regrowth of denuded endothelium. Anthocyanins isolated from black soybean seed coat enhance wound healing, through cytoprotective effect, enhance angiogenesis, and exert anti-inflammatory effect . Chang et al., (2006) have shown that anthocyanin-rich extract of Hibiscus inhibited LDL oxidation and OxLDL-mediated macrophage apoptosis .
mRNA analysis of experimental cells as shown in figure (7(a-d)) shows down regulation of pro-inflammatory molecules MCP-1 and IL-6 and membrane scavenger receptor LOX-1 levels when treated with OPC, whereas increased expression proinflammatory molecules supports the accumulation of ROS, increased adhesion of monocytes, increased expression of endothelial scavenger receptor LOX-1. L-Selectin is a type I, membrane cell adhesion molecules that is expressed on the surface of most circulating leukocytes. The function of L-selectin on the leukocytes to leave the bloodstream, make random contacts and tether to activate endothelial cell layer allows leukocytes to encounter their target antigen in inflamed tissues. Endothelial ligands of L-Selectin are GlyCAM-1, CD34, Sgp200 and PCLP, which enhances L-selectin expression on the monocytes supports tethering and rolling along vascular endothelium.
Abnormal transendothelial migration of monocytes initiated by accumulation of OxLDL in the subendothelial layer of the blood vessels is a well-known factor, which elicits atherosclerosis . This transmigratory process involves several steps including slow rolling, adhesion strengthening, crawling, paracellular and transcellular migration and transmigration through the basement membrane, which is mediated by Monocyte Chemoattractant Protein-1 (MCP-1), a chemokine that plays an important role in monocyte trafficking across the endothelial layer . Previous studies have clearly shown that MCP-1 plays a pivotal role in the monocyte migration and its further differentiation into macrophages under the induction of OxLDL . The mRNA expression of MCP-1 was found to be elevated in the OxLDL treated coculture, when compared to Group 1. This could be because of OxLDL is known to elicit the chemokine MCP-1, further responsible for the increased transmigration of monocytes, endothelial dysfunction and activation. This is in agreement with other studies, that the migration of leukocytes towards the site of inflammation is highly dependent on chemotactic effects of MCP-1. As suggested by other researchers, upregulation of MCP-1 by OxLDL, further activates LOX-1 , IL-6  and TF  expression on the endothelial cells, which directly influences the endothelial dysfunction. MCP-1 levels were found to be significantly reduced in the OPC treated group, due to their antioxidant activity. Numerous lines of evidence have favored the antioxidant activity of phytomedicine in suppressing the levels of MCP-1. Ginkgo biloba extract attenuated OxLDL-induced oxidative functional damage in endothelial cells [ ]. Another study by Ou et al. (2010) showed that EGCG also protects the OxLDL induced endothelial dysfunction via inhibiting LOX-1 mediated signaling . 6-shogaol prevents the OxLDL-induced LOX-1-mediated biological events in HUVECs, probably via its antioxidative and anti-inflammatory functions . Resveratrol inhibits the effects of Nox1 and MCP-1 expression via Akt and FoxO3 signaling pathways . Thus, treatment with OPC attenuates the MCP-1 levels and thereby limits the transendothelial monocyte migration associated with inflammation.
Studies have shown that induction of LOX-1 expression is stimulated by many factors, such as inflammatory cytokines, oxidative stress, hemodynamic stimuli and OxLDL . LOX-1 is considered as the key atherogenic factor; because under normal conditions, the expression of LOX-1 is rarely detected, whereas, upon proinflammatory and proatherogenic stimuli, LOX-1 is upregulated . Recent evidences have demonstrated that LOX-1 activation induces oxidative stress which, in turn, stimulates more LOX-1 expression, suggesting a direct relationship between oxidative stress and LOX-1 expression . When treated with OPC there was marked decrease in the LOX-1 expression. This suppression of LOX-1, may be due to antioxidant and anti-inflammatory properties of OPC, which is, supported by other investigations on the anti-atherogenic effects of ellagic acid, EGCG, anthocyanins, 4-Oxo-Flavonoids and procyanidins, that exert their protective effects by inhibiting ROS generation, thus, attenuating CRP+OxLDL induced LOX-1 upregulation in endothelial cells .
Monocyte adhesion onto the endothelial cells further stimulates the secretion of IL-6 involved mainly in inflammation and eventual progression to atherosclerosis . This study also showed increased expression of IL-6 in the CRP+OxLDL induced group, when compared to control group. Previous studies have stated that the OxLDL induces IL-6, CRP and MCP-1 suggesting an inflammatory process and its association with calcification. OPC showed reduced CRP+OxLDL induced IL-6 mRNA expression, when compared to CPR+OxLDL induced group. From previous study, supplementation of anthocyanins had been demonstrated to modulate the expression of both IL-6 and VCAM-1. Feeding anthocyanin rich bilberry and strawberry beverages to human participants with elevated risk of CVD reported reduced plasma concentrations of IL-6 and C-reactive protein . The effect of Cyanidins 3 Glycoside metabolites on OxLDL induced IL-6 production in vitro suggests its anti-inflammatory effect .
ROS leads to disruption of mitochondrial membrane potential (Δψ), in general this process is considered as an early step toward cell death. The FACS analysis of JC-1 dye in control and experimental groups (Figure 8(a)). These results suggested that JC-1 aggregates were seen in cells treated with OPC, whereas JC-1 monomers were seen in cells treated with CRP+OxLDL. The ability of OxLDL to stimulate entry of cells into cell cycle arrest was analysed by flow cytometry. The anti-apoptotic effect of OPC on endothelial cells was evaluated in two separate sets of experiments, one with co-culture and the other just the endothelial cells alone. The co-culture model was analysed for cell cycle arrest upon OxLDL stimulation. Figure: 8(b) showed a substantial decrease in the proportion of cells in G0/G1 phase and increased cell death. Therefore, OxLDL induction demonstrates cell cycle arrest and inhibition of growth. Surprisingly, OPC treated group also showed G0/G1 arrest and showed increased cell death as compared to OxLDL treated group, while in all other experiments of this study, OPC had shown promising cytoprotective effect. This vividly different result was observed only for the co-culture induced with CRP+OxLDL and treated with OPC.
The possible intervention of OPC against CRP+ OXLDL was summarized in figure (9). Therefore, from the above results and discussion, this study strongly supports the anti-oxidant, anti-angiogenic and anti- inflammatory effects of OPC against CRP+OxLDL induced endothelial dysfunction. Hence, OPC could be able intervene the inflammatory phase triggered upon CRP and OXLDL induction and thereby OPC may be potent cardioprotective agent.