Raised leptin levels have been reported in placentae and serum of women with elevated blood pressure and proteinuria during pregnancy. The role of leptin in this however remains unclear. ACE2 is a new member in RAAS, which is reported to have hypotensive and anti-inflammatory effect and its suppression leads to increased blood pressure and endothelial activation. Therefore, this study investigated the effect of leptin and xanthenone (ACE2 activator) administration on systolic blood pressure (SBP), proteinuria and serum markers of endothelial activation during pregnancy in Sprague-Dawley rats. Eighty female Sprague-Dawley rats, aged 12- 13 weeks were randomised into 10 groups, Group 1 acted as a control non-pregnant group and given saline (NSNP)

Ibrahim, Hisham Saleh

Universiti Teknologi MARA Institutional Repository


I n s t i t u t e  o f  G r a d u a t e  S t u d i e s  ( I G S )
* (MS) = Main Supervisor       (CS) = Co Supervisor
20
27
Faculty of Medicine
The development of atherosclerotic plaques is a multistep process 
involving changes in blood lipid composition, dysfunction of the 
endothelium, and infiltration of inflammatory cells. Cellular and molecular 
studies revealed enhanced expressions of several genes in development 
of atherosclerosis. This thesis aimed to investigate whether changed 
expressions of endothelial surface genes (VCAM, ICAM, and selectins), 
MCP-1, MMPs, and tissue inhibitor of MMPs (TIMPs) are associated with 
the underlying changes of the endothelium and subendothelial space in 
the development of atherosclerosis. In addition, the present study also 
determined whether any novel differentially expressed gene (DEG) is 
associated with atherogenesis. Rabbits were fed with 1 % cholesterol to 
induce atherosclerosis. Blood serum was collected for lipid profile analysis. 
Aorta tissues were used to study changes in morphology, ultrastructure, 
and gene expressions. Luminal endothelial surface from rabbit aortic 
tissue was examined by scanning electron microscopy (SEM) using low 
vacuum mode. The tissue cross-sections were stained with hematoxylin 
and eosin (H&E) for microscopic observations of intimal thickening. Total 
RNA was extracted from aorta tissues for gene expressions analysis. 
Differentially expressed genes (DEG, were analyzed by Real-time 
polymerase chain reaction (PCR) and Quantigene® Plex. Annealing 
Name :
Maimunah Mustakim
Title :
Molecular Events Associated With 
Underlying Changes Of The Vascular 
Endothelium And Subendothelial 
Space During Atherogenesis In An 
Animal Model
Supervisor : 
Associate Prof. Dr. Mohamed 
Saifulaman Mohamed Said  (MS)
Prof. Dr. Nafeeza Mohd Ismail (CS)
Associate Prof. Dr. Gabriele Ruth Anisah 
Froemming (CS)
25
26
Diabetes Mellitus is notorious for its metabolic effect, acute and long 
term complications and impact on Quality of Life (QOL). Plethora of 
literature has documented the negative impact of DM on QOL. Currently, 
religion and spirituality constitute a topic of great importance to most of 
the world’s population where researchers have notably focused attention 
on the relationship between religion, spirituality and Quality of Life 
(QOL). However there is little, if none is known about the relationship of 
spirituality and diabetes-related QOL. The primary aim of this study was 
to determine factors affecting QOL among sample of patients with type 2 
diabetes mellitus attending the medical centre of National University of 
Malaysia, Kuala Lumpur, Malaysia specifically in relation to spirituality. For 
this purpose we had to translate the English version of Spiritual Wellbeing 
Scale (SWBS) into Malay language as well as validate the Malay version 
of SWBS among Malaysian general population at Klang Valley and thence 
we proceeded to measure diabetes-related QOL among diabetic patients at 
the National University of Malaysia Teaching Hospital. Two questionnaires 
were used in this study; the Malay version of Spiritual Wellbeing Scale and 
the Audit of Diabetes Dependent Quality of Life (ADDQOL-18). The Malay 
SWBS is made of 20 items rated on 6 point Likert scale. The ADDQOL-18 
Raised leptin levels have been reported in placentae and serum of women 
with elevated blood pressure and proteinuria during pregnancy. The role 
of leptin in this however remains unclear. ACE2 is a new member in RAAS, 
which is reported to have hypotensive and anti-inflammatory effect and its 
suppression leads to increased blood pressure and endothelial activation. 
Therefore, this study investigated the effect of leptin and xanthenone 
(ACE2 activator) administration on systolic blood pressure (SBP), 
proteinuria and serum markers of endothelial activation during pregnancy 
in Sprague-Dawley rats. Eighty female Sprague-Dawley rats, aged 12-
13 weeks were randomised into 10 groups, Group 1 acted as a control 
non-pregnant group and given saline (NSNP). Group 2, control pregnant 
rats, was given saline (NSP), group 3 was given 60 μg / kg /day of leptin 
starting from the 1st day of pregnancy (LD1-60), group 4, was given 60 μg 
/ kg /day of leptin starting from the 10th day of pregnancy (LD10), group 5, 
given leptin from day 16 of pregnancy (LD16). Group 6 (L14D-60), given 
Name :
Aqil Mohammad Daher
Title :
Determinants Of Health Related 
Quality Of Life Among Type 2 Diabetic 
Patients Attending National University 
Of Malaysia Medical Centre
Supervisor : 
Prof. Dr. Syed Hassan Syed Al Ahmad 
Mashoor  (MS)
Prof. Dr. Than Winn (CS)
Prof. Dr. Norazmi Kamaruddin (CS)
Name :
Hisham Saleh Ibrahim
Title :
Effect Of Exogenous Leptin On Blood 
Pressure, Urinary Protein Excresion, 
Endothelial Activation And Ace2 
Expression During Pregnancy In The 
Rat
Supervisor : 
Prof. Dr. Harbinder Jeet Singh A/L 
Gurcharan Singh (MS)
Dr. Nor Ashikin Mohamed Noor Khan (CS)
Associate Prof. Dr. Gabriele Ruth Anisah 
Froemming (CS)
L e a d i n g  Y o u  T o  G r e a t e r  H e i g h t s ,  D e g r e e  b y  D e g r e e 21
control primer (ACP)-based GeneFishing™ PCR was 
used to analyze differentially expressed unknown genes. 
The DNA fragment from DEG was cloned, sequenced, and 
validated by Real-time PCR. Presence of highly expressed 
MMP genes in the intimal thickening of atherosclerotic 
tissues was detected using immunohistochemistry (IHC) 
staining. Lipid profiles obtained from rabbits fed with 1 
% cholesterol showed highly significant difference (p 
< 0.001) in total cholesterol and low density lipoprotein 
(LDL) while terminating the study at week-2 and week-8. 
Ultrastructural observations of the aortic luminal surface 
by low vacuum mode SEM showed changes from normal 
regular smooth intact endothelium to irregular luminal 
surface including endothelial swelling and formation of 
‘craters’ on the endothelial surface. In the present study, 
we examined the aorta tissues much closer to its natural 
conditions using a preparation not subjected to critical 
drying point and heavy metal coating. Ultrastructural 
changes of the luminal surface in atherogenesis indicate 
dysfunction of the endothelium. Higher expression of 
VCAM, P-selectin, E-selectin, and chemokine (MCP-
1) might influence structural integrity of the luminal 
endothelium. H&E stained aorta tissues exhibited 
discernible intimal thickening at week-8 of atherogenesis; 
the tissues were found to be consisted of abundant foam 
cells. MMPs and TIMPs showed different expression 
profiles in Real-time PCR and Quantigene® Plex assays. 
Highest MMP-12 expression was detected by both assays 
at week-8 atherogenesis. IHC staining of the foam cells 
detected expressions of MMP -1, -3, and -12 in week-8 
aorta tissues. We identified DEG detected from ACP-41 as 
cathepsin B gene; it was highly expressed at week-8 and 
week-12 of atherogenesis. Based on the findings of the 
present study, we can conclude that loss of endothelium 
integrity is associated with higher expressions of several 
types of endothelial surface genes. Additionally, we 
also found that intimal thickening was associated with 
differential expression profiles of MMPs and TIMPs 
genes. We also identified Cathepsin B as proatherogenic. 
is composed of two overview items and 18 life domains 
which are rated for both impact of diabetes and importance 
to diabetic patients. Data for the validation phase were 
collected from 623 Malaysians from three main ethnic 
groups in Klang valley by trained enumerators. Data for 
measuring diabetes-related QOL was collected by trained 
research assistant from 256 patients with type 2 DM who 
were attending diabetes clinic at the National University 
of Malaysia Medical Centre. Descriptive statistics were 
produced for all study variables. Exploratory factor 
analysis with promax rotation was used to explore the 
factor structure of the Malay SWBS and determine the 
reliability coefficient. Stepwise multiple linear regression 
was used to identify factors associated with diabetes 
QOL. The results of the study showed an equivalent 
translated version of the Malay SWBS .The validity of the 
Malay SWBS was ascertained with the findings of three 
factors model explaning 59.70% of the total variance, 
and a reliability coefficient of more than 0.7. Diabetic 
leptin approximately 14 days before pregnancy, Group 7 
was non-pregnant rats receiving leptin for a period of 20 
days (LNP). Group 8, given 60 μg / kg /day of leptin with 
600 μg / kg /day of xanthenone (XNT), an ACE2 stimulant 
from day 1 of pregnancy (L+ACE2a), while group 9 was 
given 600 μg / kg /day of XNT alone starting from day 1 of 
pregnancy (ACE2a). Group 10 was given 120 μg / kg /day 
of Leptin starting from day 1 of pregnancy (LD1-120). SBP, 
serum ACE, ACE2, endothelin-1, E-selectin and ICAM-1 
levels were estimated. ACE2, endothelin-1, E-selectin and 
ICAM-1 gene expressions were determined in the kidney 
and aorta. Data were analysed using ANOVA and post-hoc 
analysis, data are presented as mean ± S.E.M. Compared 
to group 1, SBP was higher in the leptin only treated group 
(P<0.001) and lower in rats treated with xanthenone alone 
patients had high proportion of diabetes complications, 
poor glycemic control, hypertension and obesity. The 
QOL among diabetic patients in this study was negatively 
affected. Multiple linear regression showed that glycaemic 
control (HbA1c), diabetes worry, use of insulin, more than 
10 years’ duration of diabetes, neuropathy and retinopathy 
were associated with poor quality of life, whereas being 
satisfied with waiting time for consultation and being 
spiritually affiliated , were associated with better QOL. 
We concluded that the QOL among the study sample 
was negatively affected by diabetes. Measures to reduce 
diabetes complications through better glycemic control 
and well-tolerated treatment modality, and reducing 
waiting time would go a long way to improve the quality 
of life. The positive relation of spirituality to QOL among 
diabetic patients opens new vista for further research in 
the field.
(P< 0.01). ACE2 activity and expression were lower in 
leptin only treated rats (P<0.05). Urine protein excretion, 
serum endothelin-1, serum E-selectin, and ICAM-1 levels 
were significantly higher than controls in leptin only 
treated rats (P<0.05) but not in the others. It seems, leptin 
administration during pregnancy significantly increases 
SBP, urinary protein excretion, levels and expression of 
markers of endothelial activation, but decreases the level 
and expression of ACE2. These effects are prevented 
by xanthenone, implicating the role of ACE2 in leptin-
induced raised blood pressure and proteinuria during 
pregnancy. However, further studies are required to 
examine the underlying mechanism responsible for this 
and its relevance to preeclampsia in humans. 


Effect of exogenous leptin on blood pressure, urinary protein excresion, endothelial activation and ace2 expression during pregnancy in the rat / Hisham Saleh Ibrahim

http://ir.uitm.edu.my/19615/1/ABS_HISHAM%20SALEH%20IBRAHIM%20TDRA%20VOL%206%20IGS_14.pdf

Effect of exogenous leptin on blood pressure, urinary protein excresion, endothelial activation and ace2 expression during pregnancy in the rat / Hisham Saleh Ibrahim

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