Clinical trial NCT01420328
Conditions: Inflammation
Sponsor: University at Buffalo
Start date: 2011-05-01; Completion date: 2016-07-01
Enrollment: 20 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study focuses on the use of Vytorin to study inflammatory markers in subjects with normal cholesterol.

Detailed description:
Following the first demonstration by our group that macronutrient (glucose, cream and a high fat high carbohydrate meal) intake results in increased ROS generation and oxidative stress at the cellular and molecular level, the investigators have now shown in our preliminary data that cream intake induces comprehensive inflammation as reflected in increased intranuclear NFkB binding, decreased IkBα expression, increased expression of IL-1β, IL-12, TNFα and other pro-inflammatory mediators. While carrying out these experiments, the investigators asked whether cream intake was associated with an uptake of lipid by peripheral blood mononuclear cells (MNC). Indeed, there was a significant increase in intracellular lipid which was visualized as intracellular lipid droplets. The increase in intracellular lipid droplets was associated with an increase in intracellular superoxide generation; the expression of CD68, a marker for macrophages; and PECAM, the adhesion molecule which mediates trans- endothelial transfer of leucocytes. The investigators also found that the lipid fractions to increase were cholesterol ester, triglyceride and fatty acids. In view of the tantalizing observation that the lipid droplet laden MNC appeared to be monocytes, looked like foam cells and the fact that CD68 expression had increased, there is a possibility that foam cells may be formed in peripheral circulation by monocytes after a lipid rich meal. This simple model of foam cell formation also lends itself for the study of the effect of various lipid lowering drugs. Our investigation will be the first to study this novel paradigm. The investigators plan to study the effect of a cholesterol lowering agent, Vytorin (simvastatin and ezetimibe), on intracellular lipid in MNC, expression of CD68 and PECAM, ROS generation and inflammation in obese subjects. This investigation may provide an additional mechanism of action by which these drugs may reduce atherosclerosis.

Arms: arm 1: Obese subjects treated with placebo for 6 weeks arm 2: Obese subjects treated with Vytorin for 6 weeks
Interventions: intervention 1: Vytorin intervention 2: Placebo
Intervention descriptions: intervention 1: Simvastatin 40 mg and Ezetimibe 10 mg daily combination pill (Vytorin) for 6 weeks intervention 2: Placebo treatment for 6 weeks

Protocol documents:
Prot_SAP_000.pdf: 
The Effect of Vytorin on Intracellular Lipid and Inflammation in Obese Subjects  
 
Version: 03/27/2012 
 
 
 
INVESTIGATOR: 
 
Paresh Dandona, M.D., Ph.D 
 
 
  
 
SUB-INVESTIGATOR(S):  
Ajay Chaudhuri, M.D.  
 
 
 
 
 
 
Sandeep Dhindsa, MD. 
 
 
 
 
 
Antonio Makdissi MD 
 
 
 
 
 
Reema Patel MD 
 
 
 
 
 
Husam Ghanim, PhD 
 
 
Study Site: 
Diabetes-Endocrinology Center of Western New York 
Division of Endocrinology, Diabetes and Metabolism 
115 Flint Road 
Amherst, NY 14221 
Phone Number: 716-626-7998  or 716-626-7999 
 
 
*Corresponding Investigator – pdandona@kaleidahealth.org 
 
 
 
SOURCE OF SUPPORT: Merck Pharmaceuticals 
 
 
NCT01420328 
 


Abstract 
 
Following the  first  demonstration  by  our  group  that  macronutrient  (glucose,  cream  and  a  high  fat  high 
carbohydrate meal) intake results in increased ROS generation and oxidative stress at the cellular and molecular 
level, we have now shown in our preliminary data that cream intake induces comprehensive inflammation as 
reflected in increased intranuclear NFB binding, decreased IBα expression, increased expression of IL-1β, 
IL-12, TNFα and other pro-inflammatory mediators. While carrying out these experiments, we asked whether 
cream intake was associated with an uptake of lipid by peripheral blood mononuclear cells  (MNC). Indeed, 
there was a significant increase in intracellular lipid which was visualized as intracellular lipid droplets. The 
increase in intracellular lipid droplets was associated with an increase in intracellular superoxide generation; the 
expression of CD68,  a  marker for macrophages; and PECAM, the adhesion molecule which mediates trans- 
endothelial transfer of leucocytes. We also found that the lipid fractions to increase were cholesterol ester, 
triglyceride and fatty acids. In view of the tantalizing observation that the lipid droplet laden MNC appeared to 
be monocytes, looked like foam cells and the fact that CD68 expression had increased, there is a possibility that 
foam cells may be formed in peripheral circulation by monocytes after a lipid rich meal. This simple model of 
foam cell formation also lends itself for the study of the effect of various lipid lowering drugs. Our investigation 
will be the first to  study this novel paradigm. We plan to study the effect of a cholesterol lowering agent, 
Vytorin (simvastatin and ezetimibe), on  intracellular lipid in MNC, expression of CD68 and PECAM, ROS 
generation and inflammation in obese subjects. This investigation may provide an additional mechanism of 
action by which these drugs may reduce atherosclerosis. 
 
 
 
SPECIFIC AIMS 
Our recent work shows that following the intake of cream there is an increase in intracytoplasmic lipid and 
evidence of cellular  inflammation in mononuclear cells (MNC) and monocytes. The main objective of this 
research is to investigate the effect of Vytorin (simvastatin and ezetimibe) on intracellular lipid accumulation 
and inflammatory mechanisms at the cellular and molecular level in the monocytes of humans, in vivo. 
 
Hypothesis 1 (primary objective): Vytorin (simvastatin and ezetimibe) can decrease the accumulation of 
lipids in the circulating monocytes of obese subjects. 
Aim 1.1: To study the effect of dairy cream (33 g) intake on the accumulation of cholesterol esters, cholesterol, 
triglycerides, fatty  acids and phospholipids in the monocytes of obese subjects with LDL ≥100mg/dl before 
after 6 weeks of therapy with Vytorin or placebo. 
Aim 1.2: To investigate the effect on cream intake on monocyte activation markers including CD68 and 
PECAM before and after 6 weeks of above mentioned drugs. 
 
Hypothesis 2 (secondary objective): Dairy cream intake causes an acute oxidative and inflammatory response 
at the cellular and molecular level. This inflammatory response can be decreased after therapy with Vytorin . 
Aim 2.1: To measure the activity and the expression of pro-inflammatory transcription factor NF-B and its 
activating proteins, IB Kinase  (IKK); and the expression of pro-inflammatory NF-B-regulated genes and 
proteins including IL-1β, IL-12, TNFα in normal subjects after the intake of dairy  cream before and after 
Vytorin or placebo for 6 weeks on obese subjects. 
Aim 2.2: To measure the oxidative stress in MNCs induced by cream challenge before and after 6 weeks of 
Vytorin. 
 
Hypothesis 3 (secondary objective): The intake of cream induces an increase in the expression of toll like 
receptor and the mediators of TLR signal transduction, and suppressor of cytokine signaling (SOCS)-3 in MNC 
and monocytes. This response can be decreased after therapy with above mentioned drugs. 


Change in NFkB binding (%) 
Change in IKK kinase activity (%) 
Aim 3.1: To measure the expression of TLR-2, TLR-4, MyD88, SOCS-3 and TRIF in MNC and monocytes 
after cream intake in obese subjects before and after Vytorin or placebo for 6 weeks. 
 
Background 
We have previously demonstrated that the intake of macronutrients including glucose, cream and a high 
fat high  carbohydrate (HFHC) fast food meal induces oxidative stress as reflected in an increase in ROS 
generation and an increase in plasma TBARS concentration(1). Furthermore, there is an increase in intranuclear 
NFκB binding and a decrease in IκBα, with an  increase in IKKα and IKKβ(2). Clearly, therefore, these 
macronutrients  induce  post  prandial  oxidative  stress  and  inflammation.  These  observations  have  potential 
implications in terms of the relationships between macronutrient intake and atherogenesis since post prandial 
glycemia and lipemia have been implicated in this process. In this respect, it is also important that the oxidative 
stress and inflammation induced by a HFHC meal is of a greater magnitude and lasts for longer in the obese 
than in normal subjects(2). In addition, our work has also shown that the obese start with a higher level of basal 
(fasting) oxidative and inflammatory stress than normal subjects. Since T2D patients have higher basal levels of 
oxidative and inflammatory stress, it is likely they will respond to macronutrient challenge with greater levels of 
oxidative and inflammatory stress. In this respect it is also relevant that equicaloric meals composed of fruit and 
fiber do not induce oxidative and inflammatory stress. Similarly, orange juice and alcohol do not induce either 
oxidative or inflammatory stress(3). 
Our experiments with cream have shown that its intake induces not only an increase in ROS generation 
but also lipid peroxidation products in plasma (TBARS). Our recent data show that cream intake induces an 
increase in NFκB binding, IKKα and IKKβ activity (Figure 1) and an increase in the expression of several pro- 
inflammatory cytokines including IL-1, IL-12 and IL-18 (Figure 2). It also induces an increase in another pro- 
inflammatory transcription factor, Egr-1, and c-fos (Figure 3). c-fos is a component of the transcription factor, 
AP-1 which regulates the expression of matrix metallo-proteinases (MMPs). Indeed, MMP-9 increases after 
cream intake and a HFHC meal (2). The pro-inflammatory effect of cream lasts for more than 3h. 
IKK kinase activity 
 
25 0 
 
22 5 
 
20 0 
 
17 5 
 
15 0 
 
C rea m 
W a ter 
 
*# 
 
*# 
180 
Cream challenge 
Water challenge 
 
160 
 
 
140 
* + 
 
120 
 
12 5 
100 
 
10 0 
 
75 
 
50 
 
 
 
 
 
0 
1 
2 
3 
 
Tim e (h o u rs) 
 
80 
 
 
60 
 
 
40 
0 
1 
2 
3 
A 
B 
Time (in hours) 
 
Figure 1: Percent change in NFB (A) and IKK () activities in MNC following cream intake. N=12, 
* P<0.05 by One Way Repeated Measures ANOVA followed by Holm-Sidak method for comparison 
versus the baseline, #  P<0.05 by Two Way Repeated Measures ANOVA for comparison versus control 
group. 


Change in serum PAI-1(%) 
Change in plasma IL-18 (%) 
Change in serum ICAM-1(%) 
Change in serum MMP-9 (%) 
 
 
 
 
16 5 
 
15 0 
 
P las m a In te rleukin-1 8 
 
 
 
 
Cre a m Ch a lle n g e 
W a te r C h a lle n g e 
P lasm a sIC A M -1 
 
14 0 
Cre a m Ch a lle n g e 
W a te r C h a lle n g e 
*  + 
13 0 
 
 
13 5 
 
12 0 
 
*+ 
*+ 
*+ 
12 0 
 
+ 
 
11 0 
 
10 5 
 
90 
 
 
10 0 
 
75 
90  
 
60 
 
 
0 
1 
2 
3 
 
Tim e (in ho urs ) 
 
80  
0 
1 
2 
3 
 
Tim e (in ho urs ) 
 
 
S e ru m P la s m inogen A c tiv a tor Inhibito r-1 
Serum MMP-9 
 
200 
Cre a m C h a lle n g e 
W a te r C h a lle nge  
270 
Cream C hallenge 
W ater Challenge 
180  
 
160  
 
140  
 
 
 
*+ 
* 
240 
* 
 
210 
 
180 
 
120  
 
150 
 
100  
120 
 
80 
90 
 
60 
60 
 
40 
30 
 
20  
0 
1 
2 
3 
 
Tim e (in  hours ) 
0 
0 
1 
2 
3 
 
Tim e (in hours) 
 
Figure. 2: Percent change in plasma/Serum levels of various mediators: Graphical representation of values 
obtained by ELISA  for IL-18 (A), ICAM-1 (B), PAI-1 (C) and MMP-9 (D). All values are presented as 
mean±SE.  *  P<0.05  by  One  Way   Repeated  Measures  ANOVA  followed  by  Holm-Sidak  method  for 
comparison versus the baseline, +  P<0.05 by Two Way Repeated Measures ANOVA followed by Holm-Sidak 
method for comparison versus control group.


Percent Change in TLR4 mRNA Expression 
Change in EGR-1expression (%) 
Change in SOCS-3 mRNA Expression (%) 
Change in cFOS expression (%)
EGR-1 mRNA levels 
 
cFOS mRNA levels 
 
 
 
1500 
 
Cream Challenge 
Water Challenge 
 
* 
 
 
 
900 
 
 
Cream Challenge 
Water Challenge 
 
1250 
 
750 
*  + 
 
1000 
 
 
750 
600 
*  + 
* 
 
450 
 
500 
300 
* 
 
250 
 
150 
 
0 
0 
1 
2 
3 
 
Time (in hours) 
 
0 
0 
1 
2 
3 
 
Time (in hours) 
 
Figure 3ercent change in mRNA levels of Egr-1 and c-fos by RT-PCR: All values are presented as mean±SE. 
n=12. * P<0.05 by  One Way Repeated Measures ANOVA followed by Holm-Sidak method for comparison 
versus the baseline, +  P<0.05 by Two Way Repeated Measures ANOVA followed by Holm-Sidak method for 
comparison versus control group. 
 
In our most recent experiments, we have also shown that following the intake of cream the expression of 
TLR-4 (Figure  4A)  is increased both at mRNA and protein level. This increase is observed at 1h and is 
sustained for at least 3h. Similarly, there is an increase in the expression of SOCS-3 following cream intake 
both at mRNA and protein levels (Figure 1B). This combination of effects is important since it has implications 
not only in terms of post prandial inflammation but also for a potential  role in the pathogenesis of insulin 
resistance. The selective deletion of each of these two proteins leads to a protection from diet induced obesity 
related insulin resistance. 
 
 
 
180 
 
 
160 
 
 
140 
 
WTT 
CTT 
 
* 
250 
  
 
 
*# 
225 
*# 
200 
 
175 
 
*# 
Water 
Cream 
 
120 
 
 
100 
 
 
80 
 
150 
 
125 
 
100 
 
75 
 
60 
0 
1 
2 
3 
 
(Time Hr) 
 
50 
0 
1 
2 
3 
 
Time (hours) 
 
Figure 4: Percent change in mRNA levels of TLR4 and SOCS-3 by RT-PCR: All values are presented as 
mean±SE. n=12. *  P<0.05 by One Way Repeated Measures ANOVA followed by Holm-Sidak method for 
comparison versus the baseline, #  P<0.05 by Two Way Repeated Measures ANOVA followed by Holm-Sidak 
method for comparison versus control group. 


During our experiments with cream exploring its effects on oxidative and inflammatory stress, we asked 
whether its intake would increase intracellular lipid in MNC. Indeed, we were able to demonstrate an increase 
in intracellular lipid in MNC both chemically and microscopically (Figure 5-6). These dramatic changes raised 
important issues. Firstly, it was important to define  whether this increase was in monocytes, the cells which 
infiltrate the arterial intima to form foam cells by taking up lipid, and thus forming the fatty streak. The second 
was to define the type of lipid contained in these cells. We also asked whether these cells  expressed more 
CD68, the characteristic marker for macrophages and whether they expressed more of PECAM, the adhesion 
molecule which is cardinal to the process of trans-endothelial transfer of leucocytes. Indeed, there was an 
increase in the expression of CD36, CD68 and PECAM. These findings were indicative of an increase in the 
ability of these cells to take up lipid, to transform to the macrophage phenotype and to develop the key adhesion 
molecule which would potentially facilitate their trans-endothelial transfer of these cells into arterial wall. 
 
 
 
0H 
1H 
2H 
3H 
 
Figure 5: Lipid droplet accumulation in MNC after cream intake in normal subjects. Nile Red stain of MNC 
is shown before (0 hour) and after (1, 2 and 3 hours) lipid intake. At the indicated times, cells were fixed and 
stained for lipid. This experiment was performed 6 times. Peak lipid accumulation was at 2h. Results from 
one experiment are shown. 
Time (hours) 
 
Control  0h 
1h 
2h 
3h 
 
Cholesterol Esters 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Triglycerides 
 
FFA 
Cholesterol 
Phospholipids 
 
Figure. 6: Intracellular 
accumulation of lipids in the 
MNC analyzed by Thin Layer 
Chromatography (TLC): A 
representative TLC of lipids 
extracted from the MNC at 
the baseline and following 
cream challenge in one of the 
subjects. Lane 1 is the pooled 
control sample, lane 2 
represents intracellular lipids 
at the baseline and lanes 3, 4 
and 5 represent intracellular 
lipids at 1, 2 and 3 hours 
following the cream intake. 


Our most recent experiments have also shown that following cream intake, monocytes separated from 
the MNC by magnetic beads coated with CD14 antibody show lipid loading, inflammation and an increase in 
the expression of CD68 and PECAM (Figure 7). The non-monocytic fraction of MNC does not demonstrate 
significant lipid intake. Furthermore, the lipid laden monocytes have the typical appearance of the foam cell. 
We are not certain of the fate of this cell in circulation and whether one important destination for this cell is the 
atherosclerotic plaque (MCP-1 and CCR-2). This needs to be investigated further. However, in the mean time, 
the possibility that this ‘foam cell’ formed after the intake of cream and possibly other fatty meals, is a potential 
resident of the atherosclerotic plaque has to be entertained. After all, the concept that the uptake of lipid by 
monocyte/macrophages occurs only in the arterial wall after the arrival of both the cell and LDL-C in the 
arterial intima, is a product of a series of experiments conducted, in vitro, followed by a series of  hypotheses to 
define a potential mechanism. Our  data  do not exclude the possibility that lipid may be taken up in the 
atherosclerotic plaque. However, they seriously raise the  possibility that the foam cell may be formed post 
prandially in the circulation. 
0H 
2H 
5H 
 
 
 
 
 
 
A) 
Nile Red 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
B) 
CD68 
 
 
 
 
 
 
 
 
 
 
 
 
C) 
PECAM 
 
 
 
 
 
 
Figure 7: Lipid accumulation and activation of Monocytes before and at 2H and 5H following cream intake in 
normal  subjects.  A)  Nile  Red  stain  of  monocytes,  B)  immunohistochemistry  staining  of  CD68  and  C) 
immunohistochemistry staining for PECAM. Data representative of N=8. 


Cardiovascular  disease  is  currently  the  leading  cause  of  death  in  the  developed  countries  (4). 
Atherosclerosis is the most important cause of cardiovascular disease. It is a chronic inflammation of the arterial 
wall.  Vascular  disease,  including  in   the  heart  and  brain,  is  associated  with  monocyte  activation  and 
differentiation to macrophages which infiltrate into the intima  of arteries. Activated monocytes/macrophages 
are capable of uptaking native and modified lipids from the surrounding matrix and  transform to foam cells 
(lipid-laden macrophages). Aggregates of foam cells form fatty streaks which evolve to a more  complex 
atherosclerotic lesion which eventually leads to clinical manifestations. This process involves the participation 
of many cell types including leucocytes, endothelial cells and smooth muscle cells secreting many compounds 
and enzymes that participate in the progression of the atheroma. 
The current screening tests for vascular disease are not sensitive enough to detect vascular disease in its 
early  stages.  Most  of  the  current  technology  including  EKG  stress  test,  thallium  stress  test,  ultrasound, 
angiography, CT scans and MRI are not capable of detecting micro-atherosclerotic lesions or evaluate foam cell 
formation, the crucial step in atherogenesis. Thus, an early  and direct screening test is needed to identify 
subjects  with  risk  of  developing  vascular  disease  years  earlier  than  other   technologies  so  the  proper 
prevention/treatment programs can be initiated. Plasma lipid levels are one of the risk markers for  vascular 
disease since higher plasma lipids, especially low-density lipoprotein cholesterol (LDL-C), lead to increased 
lipid  infiltration to the intima where it will be uptaken by activated monocytes/macrophages and begins the 
transformation to foam cells. Although low-density lipoprotein cholesterol (LDL-C) remains the primary target 
for vascular disease prevention, many individuals who have vascular disease do not have substantially elevated 
LDL-C. This indicates that plasma lipids alone do not necessary reflect increased foam cell formation in the 
intima and therefore, atherosclerosis. Thus, there is a need for other  biomarkers that can reflect the events 
occurring in the intima and is crucial for early detection and prevention of vascular disease. 
Postprandial lipemia, a state characterized by increased triglycerides and triglyceride rich lipoproteins 
(TRLP) is  related  to an increased risk of atherosclerosis and cardiovascular disease (5). The mechanisms 
underlying this association are not clearly understood. Part of this association can be explained by the effect of 
TRLP on the vasculature. Vogel et.al. found that intake of a single fatty meal containing 50 g fat and 900 kcal 
impairs flow-mediated vasodilatation (6). A single high fat meal containing 50 g fat and 760 kcal stimulated 
endothelial expression of adhesion molecules in normal healthy subjects(7). Furthermore, hypertriglyceridemia 
also seems to affect haemostatic variables. TRLP induce the production of antifibrinolytic, PAI-1 from cultured 
human umbilical vein endothelial cells (8). The activity of coagulation factor VII increases in response to TRLP 
remnants (9). A few studies have examined the effect of a fatty meal or lipid particles on the leukocytes. A 
challenge with 33 g of dairy cream has been shown to induce an increase in reactive oxygen species (ROS) 
generation by MNC, similar to an  equicaloric amount of glucose (1). The incubation of macrophages with 
chylomicrons and VLDL from 15 hypertrigylceridemic subjects resulted in the conversion to cells similar to 
foam cells seen in the atherosclerotic plaque (10). But the acute effect of lipids intake on the circulating MNC 
or monocytes, in particular on intracellular lipid accumulation and inflammation is not known. 
              Inflammation at the cellular level can be described as an increase in the intranuclear binding of the 
proinflammatory transcription factor NFκB to promoters of pro-inflammatory genes like TNFα. Many different 
stimuli  have  been  shown  to  activate  NFκB,  such  as  cytokines,  oxidants,  viruses,  immune  stimuli,  and 
lipopolysaccharides (11). Activation of NFκB has also  been  implicated in atherosclerosis (12). The genes 
regulated by NFκB include cytokines (e.g. IL-1β, IL-18), chemokines (e.g. MCP-1, MIF), adhesion molecules 
(e.g. ICAM, VCAM), metalloproteinases (e.g. MMP-1, MMP-9). Other transcription factors which are involved 
in inflammation include Egr-1 and AP-1. Egr-1 is a rapidly and transiently induced by a variety of extracellular 
stimuli  related to hypoxia and vascular injury, growth factors, cytokines, and physical damage to the blood 
vessels (13-16). It induces the transcription of TNF α, ICAM-1, PAI-1, TF, TGFβ, and MMPs. Egr-1 also 
regulates tissue factor (TF) expression, which is a cell surface receptor of coagulation factor VII and initiates 
the extrinsic  pathway  of  coagulation  (15).  AP-1 modulates  transcription  of  various  inflammatory  genes 
including  MMP-9  (17).  MMP-9 hydrolyses extracellular matrix components and allows the 
spread of inflammation. MMP-9 has also been implicated in atherosclerotic plaque destabilization (18). 
Toll-like  receptors  (TLRs)  activate  intracellular  signaling  pathways  that  lead  to  the  induction  of 
inflammatory cytokine genes such as TNF alpha, IL-6, IL1 beta and IL-12 in response to binding of microbial 
and foreign products including  lipopeptides and peptidoglycans from Gram positive bacteria and endotoxin 


from gram negative bacteria. TLRs activation stimulates the recruitment of a set of intracellular TIR-domain- 
containing adaptors, including MyD88, TRIF. Recruitment of MyD88 leads to activation of MAP kinases and 
transcription factor NFκB to control the expression of inflammatory genes. It has been shown that high fat diet 
induces insulin resistant in mice in a TLR4 dependent fashion. We would like to investigate the effect of lipid 
lowering drugs on cream intake induced increase in TLRs and downstream signaling. 
Our preliminary data demonstrate that monocyte transformation to foam cell begins in the circulation in 
response to increased inflammation and oxidative stress that activates lipid uptake and/or de novo synthesis. 
These activated-lipid laden monocytes will then migrate to the subendothelium and uptake more lipids and 
progress to form the atherosclerotic lesion. This observation represents an early stage in atherogenesis and can 
be used, therefore, as an early screening test for vascular disease. Other leucocytes, including T-cells and B- 
cells, are also recruited to the injury site and participate in the process of plaque formation. Determination of 
intracellular  levels  monocytes  and  lymphocytes,  therefore,  represents  a  direct  and  early   marker  of 
monocyte/macrophage activation level and the process of foam cell formation and therefore, the risk for 
atherosclerosis and vascular disease. The current proposal provides a method for the determination of post 
cream challenge levels of intracellular lipids in monocytes as a mechanistic model for vascular disease. 
 
Significance of the Study 
1.  Clinical relevance. Since the formation of the foam cell is potentially important in atherogenesis, it 
would  be  important  to  determine  whether  drugs  that  lower  LDL  cholesterol  and  atherogenic  risk 
decrease formation of these foam cells. 
2.  Mechanistic and biological/pathological significance. The confirmation of our preliminary data in a 
larger study is important since these data provide the first evidence that the formation of the foam cell 
may occur in circulation and that the monocyte may get loaded with lipid after every fatty meal. This 
may potentially contribute to atherogenesis. This study may provide a model to study novel mechanisms 
of action of lipid lowering drugs. 
 
Study Sample: 
This will be a double blind, prospective study conducted in 20 qualifying volunteers. Subjects will be 
recruited from the hospital and university. All subjects will sign a written informed consent approved by the 
institutional review board of SUNY at Buffalo. Women and members of minority groups will be encouraged to 
participate in this study and efforts will be made to recruit subjects from these groups. 
 
Subject Assignment: 
Experiments will be carried out in obese subjects. There will be 2 groups in the study:- 
Group 1 will receive Simvastatin 40 mg and Ezetimibe 10 mg daily combination pill (Vytorin) for 6 weeks 
Group 2 will receive a matching placebo pill for 6 weeks 
 
Randomization: 
  Subjects will be randomized to 2 groups with 10 patients in each using a computer generated randomization 
chart. 
 
  Group 1 will be getting vytorin 40/10 mg daily for 6 weeks 
  Group 2 will receive placebo daily for 6 weeks. 
 
Inclusion Criteria for all groups: 
• Age: 18 to 65 years of age. 
• Obese (BMI ≥30 kg/m2) 
• LDL cholesterol of ≥100 mg/dl 
• Subject will be available for duration of the study and willing to comply with all study requirements 
• Written and informed consent signed and dated 
• Not taking any vitamins or antioxidants 
 
 


Exclusion Criteria: 
• Currently using anti-hyperlipidemic therapies 
• Triglycerides >500 mg/dl. 
• Myocardial infarction, angioplasty/stent placement or coronary artery bypass surgery in the past 6 months 
• Patient on chronic use of non-steroidal anti-inflammatory drugs or steroids 
• Hepatic disease 
• Renal impairment 
• History of drug or alcohol abuse 
• Participation in any other concurrent clinical trial 
• Use of an investigational agent or therapeutic regimen within 30 days of study. 
• Smoker 
• Pregnancy 
• Premenopausal women who are not on birth control pills and have not had a hysterectomy or tubal ligation 
• Anemia with hemoglobin <12 g/dl 
 
RECRUITMENT OF SUBJECTS: 
 
The recruitment into the study will be carried out by the following methods: 
 
1. Patients for the Diabetes-Endocrinology Center of WNY Clinic 
2. Diabetes-Endocrinology Center of WNY - Research Division - Recruitment Database 
 
Accrual Rate: On an average, we plan to recruit 1 patient per week. 
 
All subjects will have completed the following procedures prior to participating in the study. 
1) Medical History; 
2) Physical Exam 
3) Informed consent 
4) Baseline lab draws to measure CBC, SMA and lipid profile. All labs will be drawn in the fasting state in the 
morning before 10am. 
 
Informed Consent  
Study process and required visits will be explained to patient in lay terms. All subjects will be asked to sign a 
consent form approved by IRB and a copy given to all subjects. 
 
STUDY PROCEDURES 
Subjects will be randomized to receive Vytorin or matching placebo for 6 weeks. 
Visit 1 at week 0: Subjects will arrive after having fasted (10 hour) overnight. A fasting blood sample will 
be taken, followed by cream challenge and blood sample will be drawn 1, 2, 3 and 5 hours after the cream 
challenge. Cream challenge: Subjects will be given 100 mL of dairy cream (gourmet heavy whipping cream, 
Land ‘O’ Lakes Inc., Arden Hills, MN) to be consumed over 10 minutes.   This preparation of dairy cream has 
33 g fat (300 Calories) per 100 mL. 
Subjects will then be given a supply of their drug to be taken once a day at bedtime. 
Visit 2 at week 3: Subject will come fasting for a short visit for a blood draw at 4 weeks. 
Visit 3 at Week 6: A fasting blood sample will be taken, followed by cream challenge same as the 1st visit 
and blood sample will be drawn at 1, 2, 3 and 5 hours after the challenge. Subjects will then be discharged from 
the study. 
 
COMPENSATION FOR PARTICIPATION: 
To help defray the costs of your participation, you will receive $150.00 for completing the entire study. In the 
event that you do not complete the entire study, you will receive $60.00 at Visit 1 and Visit 3 each, and $30 for 
Visit 2. You will not be paid for the screening visit.  . Please be aware that occasionally additional visits are 


required, for safety evaluations or to repeat blood test, and that these visits are not a part of the 3 required visits.  
There will be no additional compensation for extra visits. 
 
METHODS: 
1)  MNC and PMN isolation by Ficoll-hypaque method: Blood samples will be collected in Na-EDTA as an 
anticoagulant.  Three and a half mL of anticoagulated blood sample are carefully layered over 3.5 mL of 
PMN  medium  (Cedarlane  Laboratories,  Hornby,  ON). Samples are centrifuged  and  at  the  end  of  the 
centrifugation, two bands separate out at the top of the RBC pellet. The top band consists of MNC, while 
the bottom consists of PMN. The MNC band is harvested and washed twice with Hank's balanced salt 
solution (HBSS). This method provides yields greater than 95% pure PMN and MNC suspensions. 
2)  Monocytes isolation by magnetic beads. Monocytes will be isolated from MNC fraction by magnetic 
beads coated with CD14 antibodies ( Maltyni Biotec, Auburn, CA) . 
3)  NF-B activation: Nuclear  NF-B DNA binding activity will be measured by EMSA. DNA-binding 
protein extracts will be prepared from monocytes by high salt extraction as described by Andrews et al 
(19). NF-B gel retardation assay will be performed using double-stranded oligonucleotide containing a 
tandem repeat of the consensus sequence for the NF-B binding site is radiolabeled with -P32 by T4 kinase. 
Then, 5 
g of the nuclear extract will be mixed with the incubation buffer and the mixture is pre-incubated at 4 
C 
for 15 minutes. Labeled oligonucleotide (60,000 cpm) is added and the mixture is incubated at room 
temperature for 20 minutes. Samples will be then applied to wells of 6% non-denaturing 
polyacrylamide gel.  The gel is dried under vacuum and exposed to X-ray film. The specificity of the band 
is established by super shift EMSA using antibodies against p65 and p50 proteins. Negative and positive 
controls will be also used to confirm the specificity of the bands. 
4)  Western blotting: MNC total cell lysates are prepared as and electrophoresis and immunoblotting will be 
carried as described before (20). Polyclonal or monoclonal antibodies against TLR4 (Abcam, Cambridge, 
MA), CD68, (Transduction labs, CA), PECAM, IKK and actin (Santa Cruz Biotechnology, CA) are used 
and the membranes are developed using super signal,  chemiluminescence reagent (Pierce Chemical, IL). 
Densitometry is performed using molecular analyst software (Biorad, CA) and all values are corrected for 
loading with actin. 
5)  ROS generation measurement by chemiluminescence as an index of NADPH oxidase activation: Five 
hundred L of PMN or MNC (2 x 105  cells) will be delivered into a Chronolog LumiAggregometer cuvette. 
Luminol is then added, followed by 1.0  L of 10 mM formylmethionyl leucinyl phenylalanine (fMLP). 
Chemiluminescence is recorded for 15 minutes. In this assay system, the release of superoxide radical as 
measured by chemiluminescence, has been shown to be linearly correlated with  that measured by the 
ferricytochrome C method. The interassay coefficient of variation of this assay is 8 %. 
6)  Nile Red staining of intracellular lipids: Freshly isolated monocytes are layered on slides treated with 
poly lysine solution for 20 minutes. Fix cells with freshly prepared 3.7% formalin solution followed by 2 
PBS washes, 3 minutes each. Cover cells with freshly prepared working solution of Nile red stain (1:100 of 
stock solution in  PBS) for 5 minutes. Fluorescence microscope will be used to examine lipid stain at 
excitation  maximum  =549nm  and  emission  maximum  =  628nm.  Intracellular  Lipids  Scoring:  100 
monocytes will be examined from each sample for their  stain intensity and will be given a score (0-4) 
according to the following system: 
0 
No positive stain in cytoplasm 
1 
up to 25% of cell cytoplasm stains positive 
2 
25% - 50% 0f cell cytoplasm stains positive 
3 
50% - 75% of cell cytoplasm stains positive 
4 
all/total cell cytoplasm stains positive 
7)  Circulating pro-inflammatory mediators: Plasma MMP-9, IL-1 and IL-12 will be assayed with ELISA 
Kits from R&D systems (Minneapolis, MN). 
8)  Quantification of Pro-inflammatory gene mRNA expression in MNC and monocytes by Real-Time 
Reverse   Transcription   Polymerase   Chain   Reaction   (RT-PCR):   Total   RNA   is   isolated   using 
commercially available  RNAqueous®-4PCR Kit (Ambion, Austin,TX). The quality and quantity of the 


isolated RNA is determined before using  the  RNA. One μg of total RNA is reverse transcribed using 
Advantage RT-for-PCR Kit (Clontech, CA). Real Time RT-PCR is performed using Cepheid Smart Cycler 
(Sunnyvale, CA) in which 2 μL cDNA, 10 μL Sybergreen Master mix (Qiagen, CA) along with 0.5 μL of 
20 μM gene specific primers for IL-1β and IL-12 (Life Technologies, MD) are used. The specificity and the 
size of the PCR products are tested by adding a melt curve at the end of the amplifications and by running it 
on a 2% agarose gel. All values are normalized to actin, ubiquitin C and cyclophilin A expression. 
 
9)  DATA ANALYSIS: 
Statistical analysis will be carried out using SPSS and SigmaStat software (Jandel Scientific, CA). All data will 
be expressed as mean ± S.E of arbitrary units and percent change is calculated from the means. To evaluate 
similarity  between  the  study  groups,  baseline  values  for  subject’s  demographics  will  be  compared  using 
appropriate  parametric  and  non-parametric  tests  based  upon  the  nature  of  the  data.  To  account  for  the 
anticipated wide inter-patient variability, baseline measures will be  standardized to 100%. 
All subsequent 
values will be reported as the percent change from the baseline value.  Comparisons for each endpoint will be 
made using repeated measures ANOVA. 
The primary endpoint of the study is to evaluate the change in intracellular lipids in MNCs following cream 
challenge before  and  after Vytorin therapy for 6 weeks. Our preliminary studies have shown an increase of 
70±24% in intracellular lipids staining following cream intake in normal subjects. Assuming approximately a 
25% decrease in the cream induced intracellular lipid accumulation, a sample size of 20 subjects per group 
should provide adequate power (β = 0.8) to detect a statistically significant difference (α = 0.05), provided the 
standard deviation of the residuals is not equal to or greater than the mean difference. 
Secondary  endpoints:  We  will  measure  markers  of  monocyte  activation  including  CD68  and  PECAM 
expression as well  as the activity and the expression of pro-inflammatory transcription factor NF-B and its 
activating protein, IKK, the expression of pro-inflammatory NF-B-regulated genes, TLR-4, and SOCS-3 and 
oxidative stress in monocytes following cream challenge before and after Vytorin (simvastatin and ezetimibe). 
The decrease in intracellular lipids before and after combination of the Simvastatin and Ezetimibe will also be 
analyzed. 
 
Data Safety Monitoring Plan: 
A data and safety monitoring board is not required for this study.  The Investigator will be responsible for the 
proper conduct, data  collection and safety monitoring. Subjects will be given an adverse event diary and 
instructed to call the Investigator for any problems or changes in their physical condition or medications. Any 
serious adverse events will be reported immediately to the University IRB.  Recruitment and accrual will be 
reported to the IRB quarterly.  Annual reports regarding patient recruitment follow up, adverse events, dropout 
rates, and findings of the study will also be sent to the University IRB. 


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