The Role Of Homocysteine In The Promotion Of Atherosclerosis

Homocysteine

Discuss about the Homocysteine And Atherosclerosis: An Immunological Perspective.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the Western countries(Yusuf et al., 2001).It affects the heart and the blood vessels in the body , the diseases under this category are coronary artery diseases, stroke, heart arrhythmia, venous thrombosis etc.,  The topic in the present study is the role of homocysteine in the promotion of atherosclerosis which is primarily caused by chronic inflammation and accumulation of plaque in the arterial walls(Ilhan and Kalkanli, 2015).Atherosclerosis is initiated by many factors such as hypertension, diabetes mellitus, hypercholesterolemia, sedentary life style and  high homocysteine levels in the blood plasma (hyperhomocysteinemia; HHcy). These risk factors are affiliated with the stroke and heart attack too. A condition called hyperhomocystenemia(HHcy) caused by elevated levels of homocysteine is responsible for a number of cardiovascular diseases. Treatments for hypertension, diabetes mellitus and hypercholesterolemia were well studied and successful. However, the treatments for hyperhomocystenemia  are in need of a further investigation.

Studies such as, Norwegian Vitamin Trial (NORVIT) demonstrated that plasma homocysteine levels (≥13µmol/L which is considered as HHcy) were significantly reduced by 27% to 9.6µmol/L post 40 months of folic acid and Vitamin B₁₂ supplementation in patients with a history of myocardial infarction(Bonaa et al., 2006). However, these supplementations showed no effect in further preventing the cardiovascular complications (stroke and myocardial infraction). Excessive usage of Folic acid as a supplement showed an increased risk of cancer. Similar findings were reported in the Heart Outcomes Prevention Evaluation (HOPE) trial in which for 5 years a daily dose of vitamin B₆, B₁₂ and folic acid  was given to the patients with HHcy (≥13.2umol/L in plasma) and yet they showed an increased risk of myocardial infarction, stroke and death, despite the substantial reduction in the plasma levels of homocysteine (to 9.9µmol/L)(Lonn et al., 2006). Based on these observations, supplementations by Vitamin B₆,B₁₂ and folic acids to the patients with HHcy is not recommended due to a clear association with the promotion of atherosclerosis manifested as stroke and myocardial infarction. A study on correlation between the immune cells involved in the activation of atherosclerosis and HHcy would help establish the pathology of atherosclerosis.Thus, this review investigates the association of hyperhomocystenemia with the immune cells primarily involved in the inflammation and progression of the disease i.e, monocytes and macrophages.

Homocysteine

Homocysteine is homologue of an amino acid cysteine and differs only by a methylene(CH₂-) group. It is issynthesized in the body by the breakdown of an essential amino acid methionine found in meat, fish and dairy products. Methionine has a methyl group that is involved in DNA methylation and thus activation of genes.  As already discussed a buildup of homocysteine in the cells is cytotoxic and hence its concentration in the body is well regulated. In the mammalian cells the levels of homocysteine are regulated through the transsulfuration pathway where homocysteine is converted into cysteine (non-toxic metabolite) by an enzyme cystathionine β synthase. This enzyme requires vitamin B₆ to catalyse the reaction. Homocysteine may be directed to a second pathway a remethylation pathway where homocysteine is catalyzed by methionine synthase which requires vitamin B₁₂ to catalyse the reaction resulting in the conversion of homocysteine back into methionine. The remaining is exported to the bloodstream where most of homocysteine binds covalently to the major blood proteins albumin and globulin (produced by liver and immune system)(Hortin et al., 2006)whereas the rest is oxidized to become disulfide homocysteine or oxidizes with cysteine (forming disulfides) or circulates in the blood as free homocysteine.   

Inflammation

Any genetic defects in the enzymes, transporters or acquired factors such as constant high methionine diet influence the homocysteine regulation (transsulfuration and remethylation process) leads to the accumulation of intracellular homocysteine and an excess blood plama homocysteine.Numerous studies have determined that HHcy interacts with the inner layer of blood vessels called the endothelium and impair the function of the endothelium, resulting in endothelial cell dysfunction and uncontrollable proliferation of cells(R). Endothelial cell dysfunction is considered as the initial step of atherosclerosis.

Inflammation

Inflammation is body’s natural response towards harmful stimuli. Nitric oxide is a soluble gas produced in the endothelial cells by calcium-calmodulin-dependant enzyme known as nitric oxide synthase. HHcy condition indirectly diminishes the levels of nitric oxide gas by inhibiting an enzyme dimethylarginine dimethylaminohydrolase(DDAH) that degrades asymmetric dimethylarginine(ADMA) which is an inhibitor of nitric oxide thus with no enzyme activity there is an accumulation of ADMA. Nitric oxide is blocked by the ADMA(Stühlinger et al., 2001). In endothelial cells, Nitric oxide prevents low-density lipoprotein (LDL) oxidation(Hogg et al., 1993),  vascular smooth muscle cell proliferation (VSMC)(Jeremy et al., 1999), regulates local cellular growth(Papapetropoulos et al., 1997), immune cells’ (such as monocytes) activation(Zeiher et al., 1995) and prevents platelets from sticking to the endothelium(Riddell and Owen, 1999).The endothelial dysfunction is marked by a decrease in nitric oxide bioavailability (speculated to be caused by HHcy) within the walls of blood vessels leading to a series of events such as the accumulation of LDL and its oxidized form(Rubbo et al., 1994), followed by vasodilation, an increased permeability of vessels to the influx of immune cells, monocytes and leukocytes, proliferation of VSMC and platelet adhesion to the arterial walls. Several such cycles eventually allow the blood vessel to be inflamed and accumulation of plaque between the two layers of the endothelium and VSMC.The plaque once established grows in quantity as more materials (oxidized LDL, LDL, immune cells etc.) infiltrate it. This proceeds into the narrowing of the arterial lumen and the blood flow towards the vital organs such the heart and the brain is restricted. When not treated, atherosclerosis manifests as heart attack (restriction of blood flow to heart) or stroke (restriction of blood flow to brain) and often results in the death of the individual. Thus, it is crucial to determine how HHcy effects immune cell activation to understand the process involved in activating atherosclerosis.

Vertebrates have an efficient immune system, cells of the immune system survey body tissues to conduct and regulate immune responses. Immune cells can be broadly categorized into B-cells and T-cells. B-cells are involved in humoral immunity and produce antibodies.T-cells on the other hand are involved in the cell-mediated immunity. The main role of T lymphocytes (T cells) is to examine tissues and recognize foreign materials such as infectious pathogens or self-altered cells such as tumor cells. T cells have specialized T cell receptors that help them detect the histocompatibility complex I and II (MHCI and MHCII) molecules on the antigen presenting cells such as dendritic cells, phagocytes with abnormal protein fragments bound to them. MHCI molecules present abnormal peptides within the body at the cell surface to CD8⁺ T cells whilst MHCII molecules present the peptide at cell surface to CD4⁺ T cells  (Neefjes et al., 2011). CD4⁺ T cells participate in the immune responses through cytokine secretions that stimulate the T cell subsets termed as CD4 T helper cells and CD8⁺ T cells are involved in direct killing of the abnormal protein fragment (antigen) possessing cell (Carman and Martinelli, 2015).

Immune Cells and Atherosclerosis

In atherosclerosis, the role of the CD4 T cell helper 1 (T_h1) are pro-atherogenic, T_reg cells (also known as T regulatory or suppressor T cells) are athero-protective and the role of the other T cell subsets, T helper 2 (T_h2) and T helper 17 (T_h17) are yet to be determined as is the role of CD8⁺ T cells (Tse et al., 2013). Within the arterial wall, inflammation is initiated via the activation of monocytes/macrophages followed by T and B cell recruitment and activation.  Macrophages become source of tumor necrosis factor cytokine which promotes further recruitment of T and B cell to the initial process of atherosclerosis site (i.e. dysfunction endothelial cells)(Tse et al., 2013). In human and animal models of atherosclerosis there is evidence to suggest that T cells have a critical involvement in the development of atherosclerosis as all T cell subsets are detected in atherosclerotic plaques (Hansson and Hermansson, 2011, Libby et al., 2011).

In a murine genetic model of the atherosclerosis known as apo E^-/- was fed a normal chow diet with homocysteine (1.8g/L in drinking water) for 2 weeks demonstrated significant atherosclerosis lesions on aorta roots compared to the control group with a non-homocysteine diet (Ma et al., 2013). Under normal circumstances the lesion formation is inhibited by CD28 (Ma et al., 2013), a signaling protein expressed on CD4+ T cells (Beyersdorf et al., 2015). Within the exacerbated atherosclerotic lesion caused by HHcy there was an enhanced recruitment of CD4+ T cells, demonstrating a pathway of HHcy-induced atherosclerotic lesion formation in mice model. The same study demonstrated a markedly increased cytokine secretion such as IFN-g, TNF-a, IL-6, and IL-2 in HHcy-induced exacerbation of lesion formation. The pro-atherogenic T_h1 secrete IL-2, whereas T_h2 cells secrete IL-6 (Romagnani, 1992).

Furthermore, HHcy has been determined to activate T cells by targeting endoplasmic reticulum stress and potentiate concanavalin A-mediated T cell proliferation in murine splenic cells (Feng et al., 2016). HHcy also increases T cell adhesion to endothelial cells (Koga et al., 2002), suggesting a method of T cell infiltration into the medial layer of arterial wall to induce inflammation and activate early signs of atherosclerosis.Together, the studies described above demonstrate the role of HHcy in the inflammation of arterial wall in atherosclerosis.

In the current project, rabbit spleen cells can be treated with HHcy and all the T cell subsets activated or proliferated can be measured to determine overall effect of Hhcyon T cells during inflammation.  

Experimental Approaches to Determine the Role of Hyperhomocysteinemia in Immune Cells

Studies conducted in the late 90’s illustrated that pathophysiologically significant concentrations of homocysteine elicit nitric oxide overproduction via upregulating inducible nitric oxide synthase (iNOS) enzyme within rat aortic smooth muscle cell (Welch et al., 1998). Cytokine-activation of NF-kappa B transcriptional factors was sought to be the mechanism running iNOS-induced nitric oxide production in the above-mentioned rat aortic smooth muscle cells. Although it seems that the excess production of iNOS-induced nitric oxide should compensate the adverse effects of HHcy on the arterial wall during endothelial dysfunction, this overproduction is actually a marker in the exacerbation of atherosclerosis. This is due to iNOS being expressed mainly on macrophages upon activation/stimulation (Woo et al., 2003). On human monocytic cell (Thp1)-derived macrophages incubated with homocysteine (0.05 to 0.1mM) markedly activated iNOS-induced nitric production via NF-kappa B activation and increase phosphorylation of IkappaB alpha protein (Woo et al., 2003), suggesting a pathway for HHcy role in the inflammatory aspect of atherosclerosis.

Macrophages are the differentiated form of monocytes in the tissues surveying them for any possible foreign materials. They are an important factor in the formation of atherosclerotic lesionsat the site of an injury to the arterial walls. Monocytes recruitment is enhanced by infiltrating through the arterial wall (30 2001 hear.org). Macrophages ingest (take up/engulf)  lipids found in the wall of the arteries and once they engulfed excess they turn into nonfunctional foam cells (13, 14 2001 hrart.org). There are three subsets of macrophages CD11b,CD14 & CD16 found associated with the inflammation(Ziegler-Heitbrock, 2015). The accumulation of macrophages in the lesions produces a wide range of cytokines including monocyte chemoattractant protein-1 (MCP-1), which in turn allows a further recruitment of monocytes.The development of lesion is dependant on the transformation of monocytes into tissue macrophages and is found to be less in the M-CSF (monocyte colonystimulatingfactor) knockout mice.Vascular endothelial growth factor (VEGF) was highly expressed in the activated macrophages along with endothelial and smooth muscle cells of human coronary atherosclerotic lesions while not in a normal artery (15). Hyperhomocystinemia has been shown to possibly/might be/is excretion of VEGF in promoting atherosclerotic lesions where macrophages accumulate(R same Article hypothisisng) as tested usingELISA.

Another important factor that is involved in atherosclerosis and linked to the expression in macrophages is nuclear factor kappa B (NF-kB) which too has been detected in the endothelial and smooth muscle cells in human atherosclerotic lesions (Brand et al.1996). NF-kB has additionally been reported to be activated in macrophages when Hcy is at pathologically significant concentrations (wang et al. 2004), and likewise in hyperhomocystienemic rats (Au-Yeung et al. 2004 + 2001 heart.org). This strongly suggests that macrophages and inflammation, homocysteinemia are related with regard to the atherosclerotic lesions. Therefore highlighting the importance of each in contributing to the development of atherosclerosis.

Conclusion

In a study by (NRC canada) they investigated the effect of folic acid on Hcy-induced NF-kB activation in macrophages…..will talk about the results and superoxide (O2-)….. (refer to my note in printed paper on how to do it result+discus briefly check underlined also. in the introduction section about Hcy and folic acid as a summary and say that folic acid was suggested to inhibit Hcy induced oxidative stress and inflammatory responses in macrophages (2006 NRC Canada). In a study conducted by Trusca et al (2016) they investigated the apolipoprotein E (apoE) gene expression and signalling pathways in response to elevated Hcy levels, knowing that apoE can be produced by macrophages in response to/ responding by cholesterol metabolism (need to find Reference). They used cells and treated them with different Hcy concentrations ranging from 50µmol/L to 750µmol/L. This was tested via reverse transcription polymerase chain reaction (RT-PCR) and western plot. Where apoE expression was down regulated by a factor of two to three at Hcy concentrations of 250µmol/L to 750µmol/L, from the decrease of mRNA levels in human embryonic kidney (HEK-293) cells. However the apoE expression did not significantly change by the lower Hcyconcentrations  (100µmol/L). While From the immunoblotting data similarly the apoE was not shown to be negatively controlled by elevated Hcy.    

They also tested for the effect of elevated Hcy on apoE regulatory elements, via calcium phosphate transient transfections (Raw 264.7) and HEK-293 cells. Where it has been shown that apoE promoter decreased its activity via Hcy regardless of (ME2) in both RAW 264.7 and HEK-293 cells. Additionally they investigated the apoE promoter, downstream regulator of Hcy, through the use of specific inhibitors or a dominant negative form of IKβ. Where other transfections involved specific binding sites for NF-kB, AP-1 or nuclear factor of activated T cells (NFAT). This effect of Hcy on apoE promoter activity was opposed (counteracted) by MAPK/ERK kinase 1/2 (MEK1/2) inhibitor U0126, this has suggested the involvement of MEK1/2 in the downregulation of apoE promoter activity by Hcy. This demonstrated that Hcy induced inhibition of apoE occurred when activating NF-kB. Furthermore they demonstrated that a synthetic promoter containing three NF-kB binding sites was activated by Hcy, but Hcy have been show not to effect promoters containing AP-1 or NFAT binding sites.

After that they treated HEK-293) with 500µmol/L Hcy concentration was used to detect the binding of NF-kB p65 subunit to apoE promoter via chromatin immunoprecipitation (ChIP). While they incubated control cells with cystesine at similar concentration, and NF-kB p65 bound to DNA was incorporated with anti p65 antibody and DNA identified via PCR through using primers to amplify apoE gene at region -100/+4. From this method they revealed that following the Hcy treatment of cells the NF-kB p65 subunit is recruited to the apoE promoter. In conclusion, the Hcy stress negatively modulate apoE expression via NF-kB and MEK1/2 activation. They also suggested that the decrease of apoE expression peripherally could worsen atherosclerosis, neurodegenerative diseases and renal dysfunction.

A study conducted by Woo et al (2003) where human monocytic line and THP-1 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS), with the presence of probol 12-myristatw 12-acitate (PMA). They were incubated for 16 hours to let them differentiate into THP-1 macrophages. Next the cells were treated for 2-8 hours with an extract of ginkgo biloba (EGb) and its terpenoids, where their tested concentrations have not effected the viability of the cell as determined via trypan bue exclusion (>98%). Also they found that EGb and terpenoids have not been effected thymidine in cellular DNA highlighting no change in cell proliferation. Additionally they used NO metabolites, nitrate (NO₂^–) and nitrate (NO₃^–) to find NO production byTHP-1 macrophages as performed via nitratic oxide analyser (NOA^TM 280).  They then lysed cells in buffer containing 30 mM HEPES (pH 7.4), 0.1 mM EDTA, 10 µg/ml leupeptin, 2 µg/ml aprotonin, 1 mMphenylmethylsulfonylfluride and 0.1% (v/v) Triton X-100. This was done to measure NOS activity (iNOS activity in THP-1 macrophages).

The aim of this project is ‘To uncover the effects of homocysteine on CD40 and CD80 expression in U937 cell line.

The hypothesis considered for the study is as follows-

“Homocysteine increases CD40 in both differentiation and non-differentiation groups  and decreases CD80 in both states”.

Overall plan for U937 growth, differentiation and stimulation

General procedure for U937 passage:

U937 was grown in RPMI 1640 (Invitrogen/ Sigma Aldrich) supplemented   with   10%   fetalbovine serum (FBS), 1% Antibiotic-antimycotic solution, 2 mM L-glutamine  (Chiu et al., 2010, Jensen et al., 2015). Following this, cell lines were incubated at 37 °C in a humidified atmosphere of 5 % CO₂ (sub-cultivated 2-3 times per week if required) (Chiu et al., 2010, Jensen et al., 2015). U937 growth was carried out for the first three days. Differentiation of Pre-monocytic U937 was done from day 3 to 5by exposing to 0.1 μmol vitamin D3 for 72 hours. On day 6 and 7, U937 was stimulated by 6-24 hours.

Flow cytometry assay as was done according to the protocol. Expression of CD11b, CD14, CD16, CD40, CD80, CD83 was assayed using RPMI media alone as Negative control. On day 6, Bioplex assay for cytokine and chemokines (collecting supernatants from U937 cultures) was carried out.  Superannuation of aliquot was done and kept at -20 °C for future Bioplex assay.

Protocol for U937 experiments with Hcy:

From day 1 to 3 growth of U937 was achieved in complete RPMI 1640/ DMEM [supplemented   with   10 %   heat-inactivated FBS, 1% Antibiotic-antimycotic solution, 2 mM L-glutamine  (Chiu et al., 2010, Jensen et al., 2015)]. Incubation was done at 37 °C in humidified CO₂ incubator (5 % CO₂) (Chiu et al., 2010, Jensen et al., 2015).  On day 4, U937 was transferred into fresh complete RPMI 1640/ DMEM (supplemented   with   10 %   FBS, 2 mM L-glutamine and 1% Antibiotic-Antimycotic solution)(Jensen et al., 2015, Chiu et al., 2010). It was ensured that cells were viable. 0.1 μmol (100nM) vitamin D3waa added for 72 hours. On day 7, U937 cells were transferred complete RPMI 1640/ DMEM (supplemented   with   10 %   FBS, 2 mM L-glutamine and 1% Antibiotic-Antimycotic solution).

Three flasks were set LPS (1 ?g/ml) was used as positive control and RPMI media alone was used as negative control. Hcy (0.1 mM) was added to a 3^rd flask. For cytokine assays, after 6 and 24 h, the supernatants were harvested. Supernatants were aliquoted and stored at -20 °C for future use).

Protocol for FACS analysis of U937 Cells:

The cells were washed and re-suspended in 100μL PBS with 0.5% FBS. Cells were counted and add 5 x 10⁵ cells were added in 96 well plates. The cells were spin down and the 96-well-plate was placed in centrifuge, setting for 4 degree C. the supernatant was transferred into another labelled 96 well-plate, keeping pellet. Human FC block (1:100) was added to the pellet, and mixed and incubate for 30 minutes at 4 degree C keeping on ice. As controls only the cells were used, and isotype control with same fluorochrome for each antibody. The cells were allowed to spin in wells at 1200 rpm for 4 minutes at 4 degree C. The cells were washed once with 1 x PBS. Re-suspension of cells was done in surface antibody cocktails at correct dilution (1:200-1:500). Incubation was done for 30-45 minutes on ice in the dark. Gently tapping was done at each plate every 10 minutes to resuspend cells gently. Cells were again washed with 1 x PBS (cold), by centrifuging the 96-well-plate (1200rpm for 4 min at 4 degree C). the supernatant was discarded and re-suspended in fresh 300 µl FACS buffer and transferred to FACS tubes adjust to 300µl (total volume 300µl). Data was collected using FACS Canto (at WCHRE) while keeping on ice in the dark at all times. Analysis of collected results was done with FACS-Diva software.

As shown in table 1 of the results section, a paired t-test was performed to determine the significant differences between the groups of homocysteine and the control in CD40. The results were then graphed in figure 1 that illustrates the difference between differentiation and non-differentiation groups. The homocysteine increases CD40 in both states. However, in table 2 of the result section where the t-test was also performed to determine the differences between the groups of homocysteine and the control in CD80 that was then graphed in figure 2 as shown in the results. The figure 2 demonstrates the fluorescence intensity in percentage on U937 with the homocysteine incubation in differentiation mode (Vitamin D) and the non-differentiation mode (control). Thus, there is significant differences between both homocysteine and control in both differentiation and non-differentiation states. However, in this case the CD80 decreases in both states.

Table 1: Paired t-test between homocysteine and control in differentiation group (Vitamin D group) and between homocysteine and control in undifferentiated group (Control group) for CD40 fluorescence intensity measured in %.

CD40	Homocysteine (Vitamin D)	Control (Vitamin D)	Homocysteine (Control)	Control(Control)
Mean (%)	78.33333	59	67	48
SEM (%)	0.693889	0.333333	2.027588	0.881917

Table 2: A paired t-test between homocysteine and control in differentiation group (Vitamin D3) and between homocysteine and control in undifferentiated group (control group) for CD80 fluorescence intensity measured in %.

CD80	Homocysteine (VitaminD3)	Contol(Vitamin D)	Homocysteine (control)	Control (control)
Mean	39.75	50	39.75	50.75
Sem	0.125	0.353553	0.125	0.239357

The findings of this project are that homocysteine increases the expression of CD40 compared to control in U937 cell line differentiated (vitamin D incubation) and undifferentiated (control incubation). It is also demonstrated that homocysteine decreases CD80 expression in both differentiated and undifferentiated u937 cell lines.

It is well established that elevated levels of homocysteine increase the risk of atherosclerosis.

In the current study, the presence of higher levels of homocysteine promote the inflammatory process is tested. Severe hyperhomocysteinemia promotes bone marrow-derived and resident inflammatory monocyte differentiation and atherosclerosis in LDLr/CBS-deficient mice.

The present study shows that the unappreciated relationship between Hcy and CD40 and CD 80 is to be further researched about. This is a novel evidence of the role that Hcy plays in the pathogenesis of cardiovascular diseases. According to Tse et al., (2013) it can be stated that up-regulation of CD40 by Hcy might process through activation of NF-kB. On the other hand, products that are generated at the time of metabolism of Hcy might also have a role in the pathogenesis of atherosclerosis. In particular, cysteine had been considered as a major risk factor for cardiovascular disease. Based on previous studies it might be assumed that expression of CD40 is related to the metabolic conversion to cysteine. On the other hand, research indicates that cysteine has the high potential of driving the capability of Hcy to oxidate low density lipoprotein. This suggests that both cysteine and Hcy are notable in converging pro-oxidant pathways for up-regulating CD40. Up-regulation of CD40 through oxidant damage might be representation of a mechanism of amplification of CD40-induced proinflammatory responses (Koga et al., 2002). However, the molecular events involved in CD40 stimulation by Hcy are to be further elucidated.

Elevated levels of homocysteine are determined by genetic and dietary factors. There were previously a lack of clinical data that confirmed that a reduction in the concentrations of Hcy could lead to a reduction in clinical vascular events and mortality (Welch et al., 1998). Studies have indicated that a strong inversion exists between dietary restrictions and blood Hcy levels. Preventive strategies can be outlined for individuals who are at risk of developing artheresclerosis by lowering levels of Hcy level (Lonn et al., 2006). Such guidelines are to be developed at the earliest in this regard since there now remains strong evidence on the association between Hcy and CD40 and CD80.

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