Experimentally Induced Hyperlipidemia and Associated Atherosclerosis in the Aorta and Vascular Expression of CD 31, CD44, Beta-catenin and E Cadherin in Wistar Male Rats

Hyperlipidemia is the elevation of serum TC, TG, LDL-C and VLDL-C and decreased HDL-C levels (Rahaman et al., 2013). It is associated with cardiovascular diseases (CVD), including coronary heart disease and stroke. Hyperlipidemia is considered as the primary mediator cascade of atherosclerosis (Balakumar et al., 2007), pancreatitis, renal injury (Attia et al., 2002) and hereditary familial hypercholesterolemia (Frederick 2009). Understanding of lipid profile, alteration of aortic changes in hyperlipidemia helps in the formulation of various therapeutic agents to counter act hyperlipidemia and its associated conditions.

Vascular adhesion molecules, including selectins, vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule (ICAM-1), PECAM-1, JAMs and connexins play a role in atherosclerosis (Elena and Klaus, 2007).

Platelet-endothelial cell adhesion molecule (PECAM)-1 is a type-1 transmembrane glycoprotein of the immuno- globulin super family that is expressed on platelets, most leukocyte subsets and at endothelial cells junctions. PECAM-1 has both pro and anti-inflammatory roles in atherosclerosis (Harry et al., 2008 and Goel et al., 2008). PECAM-1 is expressed at high density at the lateral borders of EC and lower density on the surface of hematopoietic and immune cells, including macrophages, neutrophils, monocytes, mast cells, natural killer cells, lymphocytes and platelets.
 
CD44 molecule mediates through endothelial adhesion of lymphocyte and monocyte and stimulates the release of various cytokines. CD44 also acts in atherosclerotic plaque destabilization and neointimal proliferation (Protasiewicz and Adamiec 2005). Altered CD44 expression might influence the extrinsic coagulation cascade and therefore, may affect thrombus formation (Sojberg, 2007).

Cadherins are a family of cell adhesion molecules that mediate Ca²⁺ dependent hemophilic cell-cell interactions (Angst et al., 2001). Endothelial cells express E-cadherin (Lampugnani et al., 1992), N cadherin (Alexendar et al., 1993) and T cadherin (Wyder et al., 2000 and Ivanov et al., 2001), The pattern of E Cadherin expression is different in different types of atherosclerotic lesions suggest that E-cadherin is involved in the formation and progression of lesions. No E-cadherin expression was found in normal non-atherosclerotic intima (Yuri et al., 1998).
 
Catenins belong to the Armadillo family proteins. They are members of cell-adherent junctions and bind the cytosolic tail of cadherin. The cadherin-catenin complex is a target for many cell signaling pathways involved in adhesion, proliferation and cell motility (Hoschuetzky et al.,1994).

Role of the β-catenin pathway in SMC proliferation induced by oxLDL and the immunohistochemical staining of the human atherosclerotic aorta with anti-active β-catenin antibody showed an increased active β-catenin level in disrupted plaque associated with more intense positive staining in SMC layers located under the core when compared to bare expression in fibrous plaque (Aurelie et al., 2008).

Procurement of experimental animals

Male wistar rats (n=24) weighing around 200 g were procured from Sri Venkateswara Agencies, Bangalore and kept for acclimatization for one week. The rats were grouped into two (n=12 each) and housed in standard polypropylene rat cages and maintained at 25±10°C and a 12:12 h interval light/dark cycle throughout the experimental period of 90 days. Experiment was carried out in the Department of Veterinary Pathology, College of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati in the year 2017-18. The approval of the institutional animal ethical committee was obtained before the commencement of the experiment.

Source of chemicals and IHC kits
 
Cholesterol extra pure, AR grade with product code No: 77 97900 was procured from the SRL Fine Chemicals, Indian Scientific, Tirupati, Andhra Pradesh. CD31, CD44, Cadherin and Beta-catenin kits purchased from Biogenic ltd, Bangalore.

Experimental design

Hyperlipidemia was experimentally induced by feeding 1% cholesterol and 15% hydrogenated oil in 1000 g standard rat chow diet (High cholesterol diet). 
 
Clinical observations

Health condition, behavior, feed and water intake of all the rats were monitored every day and recorded clinical signs and body weight.

Hematology

Blood samples were collected in 10% EDTA at each sacrifice from all the sacrificed rats and used for the estimation of TEC, TLC, PCV by microhematocrit method Jain (1986) and Hb by Sahli’s method (Coles, 1986).

Biochemical parameters
 
At each sacrifice, blood samples from all the groups were collected into the sterile test tubes. After the blood clots formation, clear serum samples were separated without RBC and stored at 4°C. Estimation of TC, LDL-C, VLDL-C, HDL-C and TG was carried out by using commercially available biochemical kits (Auto Span Diagnostics, Bangalore).
 
Histopathology
 
Small tissue pieces of the aorta were collected in neutral buffered formalin for routine histoprocessing by paraffin embedding technique and section were stained with Haemotoxylin and Eosin [H&E] (Culling,1974).
 
Immunohistochemistry
 
Paraffin sections were cut at the 3-4 µ thickness and mounted on APES coated slides and incubated overnight at 37°C. Deparaffinized through xylene 15 min for two changes and two alcohol dips to remove xylene. Washed under running tap water for 10 min and distilled water rinsing for 5 min. Kept in the citrate buffer for 20 min (10 min at medium power and 10 min at high powering the micro oven). Cooled at room temperature and then kept in the distilled water for 5 min and in PBS for 5 min. The slides were held in the humid chamber and in the peroxidase block solution for 30 min to block the endogenous peroxidase and washed in PBS for 5 min for three changes. The power block solution was put on the tissue section for 15 min. Primary antibodies of CD31, CD44, Cadherin and β-catenin were added on the tissue sections of the aorta; then, slides were kept in room temperature for two hr, washed in PBS for 5 min for three changes. Added super-enhancer solution: The slides were kept in PBS for 5 min for three changes and added secondary antibody with HRP for 30 min. Washed in PBS for 5  min for three changes. DAB coloring reagent was prepared by adding one drop of DAB in 1 ml of a substrate. The sections were kept in the coloring reagent for 5-8 min and washed in PBS for 2 min and in tap water for 2 min. Stained with Harris haematoxylin for 1 min and washed in tap water for 5 min, dried and mounted in DPX. 
 
Statistical analysis
 
The results were analyzed statistically by performing one-way ANOVA (Snedecor and Cochran 1994).

Clinically obesity, sluggishness, poor hair coat and a non-significant increase in body weight were observed in group II rats in comparison with the control group I rats (Faheemuddin et al., 2013). Results of TEC, PCV and Hb% of group I rats was normal and non-significant throughout the experimental period. Total leukocyte count in the hyperlipidemic diet-fed group II was non-significantly higher when compared to the control group. Mohamed Anwar et al., (2008), who observed increased WBC and lymphocyte levels in rabbits that were fed with high cholesterol diet (Table 1) (Huang et al., 2001).
 


Rats on hyperlipidemic diet showed a significant (P<0.05) increase in serum TC, TG, LDL-C, VLDL-C and a significant decrease in HDL-C compared with control rats received standard basal diet throughout the experimental period  (Naiel Abbass et al., 2012; Rahaman et al., 2013). Increased serum lipid parameters in the present study might be due to hyperlipidemic diet and it indicated that the diet under trial had established hyperlipidemia in this group of rats. 

Hyperlipidemic diet significantly (P<0.05) increased the atherogenic index by the end of experiment in the hyperlipidemic diet-fed group II rats compared to control rats fed on the standard diet (Rahman et al., 2013; Harini et al., 2016). It indicates that HDL-C (Good cholesterol) is decreased in proportion to increased TC, LDL-C and it is significantly (P<0.05) evidenced by all serum biochemical parameters (Table 1).
    
Hyperlipidemic diet-fed group II rats revealed grossly pale and enlarged liver in most of the rats (Fig 1). Microscopically, microvesicular hepatic steatosis was observed predominantly and macrovesicular fatty change in a few rats (Fig 2) (Olubukula et al., 2012). Liver steatosis was absent in the control group. 
 

 


Aorta from hyperlipidemic diet-fed group II rats revealed moderate initiation of atherosclerotic lesions with degeneration of endothelial cells, subintimal lipid-laden macrophages (foam cells), slight thickening of the tunica intima with the proliferation of few SMCs. Disruption of the elastic lamina of tunica media with structural and directional changes in the myocytes was also seen in a few cases. Atheromatous plaque consisted of foam cells and SMCs and in some thrombus formation in the aorta was also evident. Similar changes were also observed in the aorta of rabbits fed with 1% cholesterol for seven weeks (Olubukula et al., 2012). Disruption of the elastic lamina of tunica media with structural and directional changes in the myocytes was also seen in a few cases. Coleman et al., (2006) Stary et al., (1995) also described thrombus formation in the aorta of humans as a type VI atherosclerotic lesion. No atherosclerotic changes were observed in the control group I rats.
         
CD 31 expression was noticed in ECs, platelets and most of the leukocyte subsets and at the endothelial cellular junctions (Fig 3) It might be due to the high level of platelets and other leukocytes in the atherosclerotic lesions and at the junction of thrombus in the hyperlipidemic diet-fed group II. CD31 is proatherogenic and expressed throughout the atheroma of the aorta (Newman 2003; Woodfin et al., 2007). PECAM-1 has both pro and anti-inflammatory roles in atherosclerosis (Harry et al., 2008; Goel et al., 2008).
 

 
         
CD 44 IHC antibody positivity was observed in the endothelial cell degeneration, subendothelial foam cell accumulation and in the thrombus also in the small blood vessels of cardiac muscle fibers with a few fat cells with attached microthrombus (Fig 4). The complete absence of CD 44 reaction was observed in the control group I. CD 44 mediates endothelial adhesion of lymphocyte and monocyte and stimulates cytokine release from macrophages and participates in the dedifferentiation of smooth muscle cells. CD44 also acts in atherosclerotic plaque destabilization and neointimal proliferation (Protasiewicz and Adamiec 2005; Sojberg 2007).
 

 
E cadherin positivity was observed in the endothelial damaged regions, foam cell accumulated areas, thrombus attached parts and the initiated atherosclerotic plaques of the large aorta and in the small cardiac vessels of the group II rats (Fig 5). Other than lesion parts of the vessel showed no reaction with E cadherin IHC antibody indicating its selective staining of lesions. Control group I revealed typical histological structures of the aorta without E cadherin positivity. Selective expression of E cadherin was observed in the atherosclerotic lesions of the group II rats indicates that it is playing a role in the initiation and progression of the atherosclerotic lesions. Yuri et al., (1998) reported that the pattern of E cadherin expression is different in different types of atherosclerotic lesions.
 

 
Subendothelial degeneration, subintimal fat vacuolation, small atheromatous plaque consisting of various necrotic cells and thrombus are majorly stained with Beta-catenin immunohistochemical antibody (Fig 6). No positive staining of β-catenin in the control group I aorta. Aurelie et al., (2008) reported modest Beta-catenin positive staining in the disrupted human atherosclerotic plaques. The cadherin-catenin complex is a target for many cell signaling pathways involved in adhesion, proliferation and cell motility (Hoschuetzky et al., 1994).
 

The hyperlipidemic diet of the present study has established the hyperlipidemia evidenced by increased TC, TG, LDL-C, VLDL-C and low HDL-C and elevated the total leukocyte count. Hepatic steatosis and degenerated endothelium with initiated atherosclerotic lesions were evidenced microscopically. CD 31, CD44, E cadherin and Beta-catenin positivity was observed in the lesions of endothelial degeneration, subendothelial lipid-laden macrophages and in the thrombus. Detection of various inflammatory and immune cell markers of atherosclerosis gives an insight into the tissue expression of these proteins, thereby the role of involved cells. It needs further quantification both in serum and at tissue levels for further understanding of these markers.

The author is thankful to the authorities of the College of Veterinary Science, Tirupati and Sri Venkateswara Veterinary University, Tirupati, for extending all the support for carrying of experiments.