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Cardiovascular diseases (CVD) are nowadays becoming growing contributors to global deaths and disease burdens, with epidemics of CVD crossing many parts of the world which are undergoing a rapid health transition(1). CVD are a major concern for human health affecting a large number of people worldwide (2, 3) . CVD are the leading killer diseases in the world accounting to 30% of the deaths of people (1). In United States of America CVD accounts for 34.3% of all deaths (4). The cost of CVD includes not only the death burden but also the costs toward health care expenditure and lost productivity from deaths and disability. This is estimated to be around $503 billion in 2010(4).
Metabolic disorders like diabetes, high cholesterol levels, obesity and insulin resistance which form the basis for Metabolic Syndrome (MS) are important factors in increasing the incidence of cardiovascular diseases(5). Is there a precise mechanism to establish a relation between metabolic syndrome and cardiovascular disease? The study of the molecular mechanisms by which CVD is increased by MS and if there are new therapeutic approaches which can be developed to target these mechanisms.. We need to explore the relationships between CVD, MS, endothelial dysfunction and endothelial nitric oxide synthase(5). The inner lining of the blood vessels is called endothelium. As long as the endothelium is healthy and producing the required quantities of endothelial nitric oxide synthase (eNOS), the risk of cardiovascular diseases is low. Any dysfunction in the vascular endothelium is the primary forecast for cardiovascular disease. The vascular endothelial function is highly dependent on the expression and function of eNOS. eNOS protein expression is determined in human endothelial cells ,collected by using angiocatheters is quantified by immunofluorescence and is compared with cultured Human Umbilical Vein Endothelial Cells (HUVEC). This data will support the function of the vascular endothelium. A correlation can be made and the outcome will direct us to predict the relationship between Metabolic Syndrome and Cardiovascular diseases.
Statement of the problem:
The inner layer of blood vessels is the endothelial cells. Endothelial dysfunction, or loss of proper endothelial function, is a primary factor for vascular diseases, and is often regarded as an important early event in the development of atherosclerosis (6).Such type of endothelial dysfunction is often seen in patients with coronary artery disease, diabetes mellitus, hypertension, hypercholesterolemia (7,8) . Endothelial dysfunction can lead to be predictive of future cardiovascular diseases (9). How endothelial function is altered with disease and the mechanisms of alteration, if present, leading to CVD need to be understood.
Therefore, if the underlying causes and the significant mechanism for developing CVD can be detected at an early stage we can prevent the further damage. This research studies the endothelial dysfunction and relation between MS and CVD, and the levels of eNOS. We need to determine the precise relations between these factors to find out earlier risk of CVD. The risks associated can be detected and proper preventive measures can be taken to reduce the incidence of cardiovascular disease in mankind. This might better aid in the early detection of CVD risks and thus allow proper preventive measures at the right time.
CVD will occur as such if there is any disturbance in the cardiovascular system but the risk factors increase the incidence of the disease.
The major risk factors for CVD:
Mean Level for Adults
Total Blood cholesterol
LDL (bad) cholesterol
HDL (good) cholesterol
(b).Overweight and Obesity: The WHO estimates that by 2015, the number of overweight people will increase to 2.3 billion, globally greater than 700 million people will be obese. This problem once encountered only in developed countries is now dramatically raising in low and middle income countries, in the urban settings. Overweight children and adolescents are more prone to increased prevalence of CVD risk factors like hypertension, hyperlipidemia and diabetes
(c).Diabetes Mellitus: A minimum of 65% of people with Diabetes Mellitus die with some form of CVD. Heart disease death rates for adults with DM are 2 to 4 times higher than for normal adults without DM.
(d).Smoking and Tobacco Use: Cigarette smoking and tobacco using people are 2 to 4 times more likely to develop CVD than people not using them. Smoking approximately doubles the risk of a person having a heart stroke.
(e).Physical Inactivity: The percentage of physical inactivity has increased in ages and groups in the United States as per the survey in 2007. The proportion of adolescents or adults using computers and watching television has increased. The proportion of adolescents engaging in physical activity has declined. Exercise alone can bring significant reduction in diastolic blood pressure, triglyceride levels and fasting glucose. 120 to 150 minutes of moderate intense activity can reduce the risk of obesity, high blood pressure, low HDL cholesterol, high triglycerides and high glucose. The risk of developing CVD with physical inactivity ranges from 1.5 to 2.4.
(f) Nutrition: Poor nutrition with more fat and Sodium and less servings of fresh vegetables and fruits and with increase in sugar sweetened beverages can increase the CVD risks. Dietary habits affect a broad range of CVD risk factors, estimation of the impact of dietary factors on cardiovascular health by considering only a limited number of pathways for example effects on lipids, blood pressure, abdominal obesity will underestimate the total impact of health. Studies conducted across and within populations showed that several nutrients, minerals, food groups and dietary patterns have an increased or decreased risk for CVD. Sufficient knowledge should be imparted to recommend nutritional interventions at population and individual levels . This knowledge should be translated into policies which promote healthy diets and discourage unhealthy diets. To achieve this there should be a coordinated action at the level of government, international organizations, civil society and responsible sections of the food industry.
(g)Family history: A positive family history of heart attack to subsequent cardiovascular health is a contributor after adjusting for age, systolic blood pressure, total cholesterol level, obesity, cigarette smoking, history of diabetes, and estrogen usage in women. In men, a positive family history of heart attack is predictive of death from all causes of cardiovascular diseases. A family history is a useful tool to identify high risk young men. .
(h).Metabolic Syndrome: Metabolic Syndrome (MS) is defined as a group of risk factors for CVD and type 2 DM. MS is also defined as a pathological phenomenon with increased CVD risk factors of metabolic origin, including visceral obesity, hypertension, dyslipidemia, impaired glucose tolerance and insulin resistance. People with any of these three diseases are defined to have MS and they are more prone (eleven times) to CVD than with people without these disease.
Fasting blood glucose
>100 mg/dl or undergoing treatment for increased glucose levels
<40 mg/dl in men or <50 mg/dl in women or undergoing treatment for reduced HDL cholesterol
>150 mg/dl or undergoing treatment for increased triglycerides
>102 cm in men and 88 cm in Women
>130mm Hg systolic or >85 mm Hg diastolic or undergoing treatment for hypertension or anti hypertension treatment with a history of hypertension
Endothelial function and dysfunction: The layer of the endothelial cells lining the blood vessels plays a functional role. A wide range of molecules produced by the endothelial cells like nitric oxide is an important compound in maintaining the homeostasis, vasodilatory, anti-inflammatory, antiplatelet formation. Inflammation and angiostasis are modulated by endothelium. Endothelial dysfunction plays a major role in CVD and MS. Endothelial dysfunction was shown to be a predictor of future CVD risks (9). When there is dysfunction there will be abnormalities in all the signaling pathways leading to pathogenesis and formation of atherosclerotic lesions. The denuded or damaged endothelium leads to the formation of atherosclerotic plaques (10, 11).
Endothelial Repair: The damaged endothelium is repaired by many processes. The endothelial progenitor cells (EPC) play a role in endothelial regeneration and new vessel formation (12). On mobilization into circulation these cells adhere and proliferate before facilitating vascular repair(13).
Role of Nitric Oxide(NO), eNOS, iNOS (inducible NOS), nNOS (neuronal NOS): The endothelium lining the blood vessels can lead to release of vasodilator substances which in turn causes smooth muscle relaxation - the main one nitric oxide(14) . Nitric oxide is a gas produced in all parts of the body. It exerts a lot of biological actions in both pathological and physiological conditions of the body (15) .NO is synthesized by three NO synthases isomers namely nNOS or type 1, iNOS or type 2, and eNOS or type 3. All the three isoforms are expressed in the cardiovascular system. Through multiple biological activities NO plays an important role in maintaining cardiovascular homeostasis (15) . NO is formed from its precursor L arginine by NO synthases with stoichiometric production of L- citrulline. It was previously thought that nNOS and eNOS are expressed in the nervous system and the vascular endothelium respectively by calcium dependant manner and iNOS is produced by stimulation of certain endotoxins or proinflammatory cytokines in a calcium independent manner. Later it was found out that nNOS and eNOS are also produced by expressional regulation and i NOS could be expressed even under physiological conditions. eNOS is targeted to signal-transducing caveolae where the enzyme with regulatory proteins. Most of the proteins are modified in CVD states. There will be increased release of NO by endothelial cells by estrogens, exercise, and dietary factors. There will be decreased levels of NO by oxidative stress, smoking, and oxidized low density lipoproteins. NO levels are reduced in vascular disease (for example diabetes and hypertension) (14) .When there is damage to arteries there will be regeneration and new endothelium is formed on arteries (12). The regenerated endothelium loses the signal for the release of NO which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction creating the way to atherosclerosis. The discovery of the complex interaction of the pathways leading to the presence and activity of eNOS may lead to understand the doubts in endothelium dependant signaling that are encountered in CVD states and may lead to detection of novel targets for therapeutic intervention. NO plays a protective role maintaining the vascular health by -decreases the smooth muscle contraction , smooth muscle proliferation and platelet aggregation , endothelin production, monocyte and platelet adhesion, expression of adhesion molecules(15) .
Figure 1: Regulation of eNOS activity and mechanisms of endothelial dysfunction at various stages. (Figure adapted from Ref.5).
Hiraoki Shimokawa et al studied individual NOS isoforms and the entire NOS system in the cardiovascular system. All types of NOS gene deficient animals, singly doubly and triply NOS deficient mice have been developed. The development of vascular lesion formation and the role of enos was studied , suggesting the vasculoprotective roles of eNOS in the pathogenesis of atherosclerotic vascular lesion formation. The role of iNOS in the vascular lesion formation is having two sides, due to oxidant and antioxidant properties of iNOS(16) . It helps in the increase of constrictive vascular remodeling. The expression of nNOS is more in the neo intima, endothelial cells and macrophages in the atherosclerotic lesions. Thus it suppresses neointimal formation. The whole of NOS has a role in the vascular system. As MS is associated with CVD, there is a role of eNOS and the whole NOS system in MS also(15) . It is possible that NOS plays an important role in preventing MS. The impairment of the NOS system is involved in the pathogenesis of the CVD. In general eNOS and nNOS have cardioprotective roles and iNOS has dual effects on the CVD system(15).
The endothelium of the blood vessels senses and responds to physiologic and pathologic stimuli by producing vasoactive substances like NO, prostacyclin and endothelins. Normal and healthy endothelium protects against plaque formation, whereas endothelial dysfunction is the main cause for atherosclerotic plaque formation. The important role of NO as the elusive endothelium derived relaxing factor was the topic of the 1998 Nobel Prize in Physiology or Medicine. eNOS serves important functions like regulation of vascular tone and blood flow suppression of vascular smooth muscle cell proliferation, modulation of leukocyte endothelial interactions and modulation of thrombosis. There are many mechanisms for endothelial dysfunction. One important feature of endothelial dysfunction is reduced bioavailability of NO in the vascular system. This may arise due to a reduction in eNOS mrna or protein expression levels. L-arginine, the substrate for eNOS, can also be a limiting factor in tissues for reduced availability of NO.
In case of diabetic patients the following pathways of the NO are impaired.
Insulin signaling: In a normal insulin signaling, glucose uptake by skeletal muscle and fat, there will be suppression of hepatic gluconeogenesis and vasodilation from increased eNOS enzymatic activity. In case of insulin resistance there is a decreased sensitivity of peripheral tissues to insulin effects. It alters the balance between the two pathways to downstream insulin signaling. There will be diminished eNOS activity low production of NO and diminished insulin mediated vasodilation.
Oxidative stress and eNOS: Oxygen production increases under oxidative stress conditions. Oxygen reacts with NO to form OONO. Thus Oxygen scavenges NO and renders it unavailable for mediating physiologic functions, including binding to soluble guanylate cyclase to stimulate vasodilation. OONO can cause direct oxidative damage and tyrosine phosphorylation of endogenous proteins thus impairing their function.(5) .
Effects on eNOS S1177 phosphorylation: eNOS is phosphorylated at serine and threonine residues. Of the potential phosphorylation sites, S1177 is the crucial regulator of the enzymatic activity of eNOS.S1177 phosphorylation results in increased electron flux through the domain and decreased calmodulin dissociation (5).This leads to eNOS being more active and produces more NO. There is evidence that eNOS S1177 phosphorylation is the crucial link between metabolism and vascular dysfunction. Animal models of diabetes, hypercholesteremia and atherosclerosis show reduced eNOS phosphorylation. Estrogens, statins and peroxisome proliferator activated receptors PPAR alpha and PPAR gamma agonists increase eNOS S1177 phosphorylation(5). Vasculoprotective signaling molecules such insulin, IGF-1, vascular endothelial growth factor, adiponectin and leptin increase eNOS S1177 phosphorylation. Laminar shear stress and flow increase S1177 phosphorylation. So, eNOS S1177 phosphorylation is an important step in regulating eNOS activity and an important target to treat endothelial dysfunction. eNOS S1177 is phosphorylated by Akt kinase, AMP kinase, protein kinase and calmodulin-dependent kinase. Shear stress, VEGF, Insulin, estrogens, statins and leptin stimulate eNOS S1177 phosphorylation by Akt kinase activation. Whereas adiponectin and resistin modulate S1177 phosphorylation by AMP kinase. This diverse signaling pathways affect multiple kinases that converge to modulate eNOS activity by S1177 phosphorylation indicating that this is the common integration point for endothelial dysfunction from various causes. When Akt kinase activity is reduced by Insulin Resistance it leads to endothelial dysfunction due to diminished eNOS phosphorylation and activity (5).
Sm mm proliferation
Figure 2: Role of eNOS S1177 phosphorylation in integrating effects of multiple mediators (Figure adapted from ref.5)
The metabolic abnormalities in the MS lead to accumulation of visceral fat in the body. White adipose tissue stores energy and also plays a role in the production of factors that affect other tissues and vasculature. These factors are resistin, IL-6, TNF alpha, whose role is to decrease eNOS S1177 phosphorylation leading to diminished eNOS activity and low levels of NO generation. Insulin resistance and visceral adiposity lead to endothelial dysfunction by reducing the NO bioavailability in the body (5).If there are any interventions in the cycle of the eNOS signaling at various stages, during insulin resistance or visceral adiposity or endothelial function, can there be a relation and the solution may lead to a discovery of the relation between MS and CVD.(5).
By measuring the eNOS levels in the endothelial cells, the relation between MS as a precursor for CVD can be detected. Unraveling the expression of this key factor (eNOS) may demonstrate a potential diagnostic and pharmacological target for treatment and detection of CVD. This will significantly reduce the global morbidity, mortality and socioeconomic burden associated with CVD.
Preliminary Data: The following data was obtained by doing lipid profile ââ‚¬"Glucose using the heparin-blood from the samples.
The measurement of total cholesterol, high density lipoprotein, triglycerides, low density lipoprotein, non hdl, glucose and ratio of total cholesterol to high density cholesterol will help us determine the lipid profile of the patients and we can determine the cardiac health of the patients by comparing these with the normal levels of these values.
The normal value of total cholesterol is less than 200 mg/dl, and I can infer from the preliminary results that one patient of the study group is having a good value and the other patients are having more total cholesterol. The normal value of HDL is 36-84 mg/dl. Three patients are below the normal range and the other six patients are at normal levels. The normal levels of Triglycerides are 90-150 mg/dl. Six patients are in the normal values and three patients are having more triglycerides. The normal levels of LDL are 64-136 mg/dl. Six patients are with normal values. The normal range of glucose levels is 70-110. Five patients are outside the normal range with one below the normal range and four above the normal range.
After doing all the lipid profiling for all the patients all the data is compared and statistical analysis is done and the values are plotted on a bar chart with error bars and the results are evaluated.
This study is based on blood collected from patients participating voluntarily.
Study population will consist of patients with either pre- cardiac diseased patients or patients with cardiac disease being treated at the Byrd Clinical Centre, Marshall University Medical Center or Cabell Huntington Hospital. The control group will consist of healthy patients without cardiac disease. The following subjects will be enrolled for study.
Males with pre-cardiac disease, females with, pre-cardiac disease, males with cardiac disease, controlled, uncomplicated females with cardiac disease, controlled, uncomplicated ,males with cardiac disease, controlled, complicated ,females with cardiac disease, controlled, complicated, males, with cardiac disease, uncontrolled, uncomplicated, females, with cardiac disease, uncontrolled, uncomplicated, males, with cardiac disease, uncontrolled, complicated, females, with cardiac disease, controlled, uncomplicated.
There will be 20 patients in each group.
Patients will be enrolled according to the following criteria:
Patients aged 18-80 with cardiac disease.
(b).Exclusion criteria: The patients with the following diseases are not included in the study.
Chronic kidney disease stage 4 and 5 (GFR<30),moderate or severe anemia (hemoglobin <10g/dl),chronic therapy with steroids or nonsteroids anti-inflammatory drugs cancer, chronic inflammatory conditions, pregnancy and nursing, history of venous thromboembolism, liver failure, AIDS, diagnosed coronary artery, cerebrovascular or peripheral arterial disease.
The treating physician will provide the research team with the contact information of patients who qualify for the research study and have expressed interest and given their permission for the release of their contact information. The study will be explained to patients verbally.
Confidentiality of the Study Data:
Each patient will be assigned a unique identification number once they are enrolled in the study. A research database will be constructed using these identification numbers to record clinical history and laboratory data. No personal identification information will be included in this database. A logbook referencing patients identities and these identification numbers will be kept separately by the principal investigator ,and only accessible by the investigators.
Schedule of Assessments: Clinical examinations and endothelial cell and blood collection will be conducted at the time of enrollment.
Study Procedures: The following procedures will be undertaken:
1 Clinical Examination:
Patients and healthy subjects will be interviewed and examined by one of the investigators at the time of enrollment. (Duration: 10 minutes).
2.Blood collection and endothelial biopsy:
Twenty (20)- guage angiocatheters (Becton Dickinson) will be inserted into a superficial forearm vein as described previously. Five J-shaped vascular guide wires (0.021-inch-diameter) will be sequentially advanced into each vein a few centimeters. Tips of the wires will be removed and washed in endothelial cell dissociation solution. The endothelial cells, which become attached to the wires, will be collected into conical tubes (2 ml). Twenty five mL of peripheral blood will be collected through the angiocatheter. Plasma will be separated by centrifugation of the blood. Twenty mL of plasma will be used for measurement of circulating miRNA. Two mL of plasma will be sent for measurement of various biomarkers. The remaining 3mL of plasma will be stored at -80C for future use.
Blood drawn from the veins may be associated with discomfort, bruising, infection at the site or formation of a blood clot. A blood clot may cause local swelling and pain that may last upto seven days. In some people the removal of blood has been shown to cause fainting.
1.Analysis of endothelial protein expression /activity:
One half of each endothelial biopsy sample (1.0 mL) will be subjected to fixation with formaldehyde and transferred to poly-L- lysine coated slides for immunofluorescent analysis of protein expression/phosphorylation. Slides will be stored at -80c. Cells will be permeabilized in PBS/0.5% Triton X 100. Nonspecific sites will be blocked with PBS containing 5% donkey serum. Cells will be incubated with anti-nitrotyrosine (Upstate Biotechnology), eNOS (Transduction Laboratories), or iNOS antibodies (Transduction Laboratories), followed by Cy3 conjugated by donkey secondary antibodies (Jackson Laboratories). Appropriate negative control slides will be generated using pre-immune IgG. Polyclonal anti-vonWillebrand factor antibodies (DAKO) will then be used , followed by FITC-conjugated secondary antibodies. Nuclei will be stained with DAPI. Between experiments variability will be standardized using reference slides of human umbilical venous endothelial cells (HUVEC) obtained from the same culture dish. Slides from patients will be stained concurrently with one slide of HUVECs. Cells will be observed under UV light with a fluorescent microscope (NIKON) using identical conditions. Nuclear and vonWillebrand factor staining will identify cells and determine their endothelial origin. Image processing and intensity quantification of the proteins will be performed using standard software and statistical methods using fluorescense and anova analysis.
2. Analysis of endothelial mRNA:
One half of each endothelial biopsy sample (1.0 mL) will be exposed to magnetic beads (Dynal Biotech) coated with a mouse monoclonal antibody specific for endothelial cells (P1H12) Chemicon International). Endothelial cells will attach to the beads by binding to the antibody and endothelial cells will be removed by three washes with saline as described elsewhere. Endothelial mRNA will be linearly amplified using a high sensitive RiboAmp HS RNA Amplification kit (Arcturus). Agilent Bioanalyser will be used to demonstrate purity and integrity of amplified RNA. The amplified RNA will then be then subjected to realtime PCR analysis of COX2, iNOS and eNOS transcripts. Beta- actin will be used as an endogenous control.
3. Analysis of circulating microRNA:
Five millilitersââ‚¬â„¢ of serum will be incubated at 560c for one hour with 0.65 mg/ml Proteinase K (Sigma P2308). The samples will be spiked with two synthetic RNAs (IDT) that will serve as positive controls. Serum will undergo acid phenol; chloroform extraction and then the RNA will be precipitated with ethanol overnight at -200c. DNase treatment will be performed to eliminate residual DNA fragments and the solution subjected to a second acid phenol; chloroform extraction. The resulting pellet will be re-suspended in double distilled water and two additional synthetic RNAs will be added to the sample as positive controls. RNA will be subjected to a polyadenylation reaction as described previously. Briefly, RNA will be incubated in the presence of poly (A) polymerase (PAP; Takara- 2180A), MnCl2, and ATP for 1h at 37c. After addition of an oligo T primer, reverse transcription will be performed on the total RNA using SuperSciprt II RT (Invitrogen). cDNA will be amplified by real time PCR using a microRNA-specific primer and a universal reverse primer complementary to the consensus 3ââ‚¬â„¢sequence of the oligo T tail.
The following procedure was conducted for the fixed endothelial cells on poly lysine coated slides:
The cells are rehydrated with 1X PBS for 10 minutes and dried. Permeabilised with freshly made 0.15% Triton X 100 for 15 min at room temperature in an incubation chamber and removed and dried. Then 0.1 M NH4Cl is added and allowed for 10 minutes at room temperature. Then the cells are blocked with blocking buffer (8ml PBS, 1ml human serum, 1 ml of 0.15% Triton X 100). Primary antibody is added at 1:50 concentration and incubated for one hour at room temperature and dried. The cells are washed twice with 1XPBST for five minutes and dried. Then secondary antibody is added at 1:250 concentration and incubated for one hour at room temperature and dried. The cells are washed twice with 1XPBST for five minutes and dried. The cells are now stained with DAPI with 1:50 in PBS for 10 min. Again washed with 1XPBS and allowed to dry thoroughly and added a cover slip with Prolong Gold and captured images on a fluorescence scope. The amount of the eNOS expressed will be quantified.
Limitations and Delimitations:
Limitations: The data about the expression of eNOS levels may be reliable or not. By knowing this expression can we predict the vascular health and the possible occurance of CVD. Is the level of eNOS expression sufficient enough to reveal the endothelial dysfunction, are the limitations we need to address. Will the immunostaining method be reliable to express the eNOS levels.
Delimitations: Will the cultured endothelial cells serve as control? to compare with the patients sample. We are not addressing questions like will the same eNOS levels can be used to detect all the various types of cardiovascular diseases like coronary heart disease, congestive heart failure, congenital heart disease , peripheral vascular disease, which have different mechanisms of action. But as endothelial dysfunction is the major factor for all these diseases we have not addressed this question.
Significance: By measuring the eNOS levels in the endothelial cells, the relation between MS as a precursor for CVD can be detected. Unraveling the expression of this key factor (eNOS) may demonstrate a potential diagnostic and pharmacological target for treatment and detection of CVD. This will significantly reduce the global morbidity, mortality and socioeconomic burden associated with CVD. There will be more improvement in the quality of lives of those patients which were detected early with cardiovascular diseases.
There will be less number of hospital visits by the people and the amount spent by the people and the government on CVD will be drastically reduced if these diseases are detected at an early stage. Awareness in the people about the risk factors and the prevalence of CVD can create a major change in the life style of people. So the significance of my study is to develop a protocol for early detection of CVD which is at a preventable stage and save not only the lives of people but also save them and the government a good amount of money.