Protective effects of certoparin sodium, a low molecular weight heparin derivative, in experimental atherosclerosis

Clinica Chimica Acta 339 (2004) 105 – 115 www.elsevier.com/locate/clinchim

Protective effects of certoparin sodium, a low molecular weight heparin derivative, in experimental atherosclerosis Perinkulam Ravi Deepa, Palaninathan Varalakshmi * Department of Medical Biochemistry, Dr. A.L. Mudaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India Received 1 July 2003; received in revised form 18 September 2003; accepted 19 September 2003

Abstract Background: The association of atherosclerosis and hypercholesterolemia is well known. Hypercholesterolemic diet-induced atherogenesis is a widely accepted experimental model that is amenable to exploration of both the disease as well as therapeutic interventions. We evaluated the role of low molecular weight heparin (LMWH) in modulating the early biochemical changes in atherogenesis. Methods: Male Wistar rats (140 F 10 g) were fed an atherogenic diet comprising of normal rat chow supplemented with 4% cholesterol, 1% cholic acid and 0.5% thiouracil (CCT diet) for 2 weeks. While one of the CCT diet-fed group served as the untreated pathologic model, the other group received LMWH (Certoparin sodium, TroparinR; 300 Ag/day/ rat s.c.) treatment, commencing on day 8 and continued for 1 week. Results: Decreased concentrations of serum albumin and increased serum urea, uric acid and creatinine concentrations were normalized by LMWH treatment. The atherogenic diet induced abnormal rise in the activities of lactate dehydrogenase, aminotransferases and alkaline phosphatase, as well as the high serum cholesterol and triglyceride concentrations were restored to near control values in the treated group. LMWH administration prevented the hypertrophic cardiac histology and fatty changes in the liver in early atherogenesis. Conclusion: The present study encapsulates the early cellular abnormalities in the heart, liver and kidney tissues of atherogenic diet fed rats. Treatment with LMWH affords considerable protection to the tissues challenged by hypercholesterolemia, evidenced by its correction of lipemia and restoration of serum and tissue indices of injury, to normalcy. LMWH intervention minimized the atherogenic diet-induced histopathological lesions in heart, liver and kidney tissues. D 2003 Elsevier B.V. All rights reserved. Keywords: Heparin; Low molecular weight heparin; Glycosaminoglycans; Atherosclerosis; Hypercholesterolemia

1. Introduction Heparins are a heterogeneous mixture of highly sulfated glycosaminoglycans with a strong negative

* Corresponding author. Tel.: +91-44-24925548; fax: +91-4424926709. E-mail address: [email protected] (P. Varalakshmi). 0009-8981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2003.09.021

surface charge. The revelation that heparin exerts functions more than just the anticoagulant effect has been a major impetus for exploring the drug for its multi-faceted biologic properties and functions at the cellular and the molecular concentration. Soon focus was directed at producing fragments of heparin, low molecular weight heparins (LMWH) that had reduced risk of bleeding for equivalent antithrombotic effect when compared with heparin therapy and were

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Table 1 Effect of LMWH on serum biochemical parameters in rats fed an atherogenic diet (compared with control groups) Serum parameters

Group I Control

Group II CCT diet

Group III Control + LMWH

Group IV CCT diet + LMWH

Urea (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl) Albumin (g/dl)

22.15 F 2.09 1.95 F 0.13 1.05 F 0.06 2.97 F 0.06

25.98 F 1.22a @ 3.67 F 0.30a * 1.28 F 0.09a # 2.10 F 0.11a *

20.79 F 2.82 1.88 F 0.10 1.07 F 0.05 3.13 F 0.10

22.04 F 1.66b @ 2.18 F 0.22b * @ 1.08 F 0.08b b * 2.98 F 0.14

Values are expressed as mean F S.D. for six animals in each group. Comparisons are made between: aGroup I and Groups II,III,IV; bGroup II and Group IV. * p < 0.001. @ p < 0.05. # p < 0.01.

otherwise comparable with standard heparin in their basic structure and biologic properties. These glycosaminoglycans consist of chains of alternating residues of D-glucosamine and an uronic acid, either gluconic acid or iduronic acid [1]. The LMWH examined in this study, certoparin sodium, is primarily indicated for clinical use in thromboembolism prophylaxis.

In the present work, an atherogenic diet protocol was been followed for 2 weeks, to study the detrimental changes in the heart, liver and kidney tissues in experimental hypercholesterolemic atherosclerosis. It has been reported that at this stage, foam cells form in the vessels [2]. High plasma cholesterol concentrations have been associated with aortic and renal lesions in the rat [3]. The biochem-

Fig. 1. Effect of LMWH treatment on serum concentrations of LDH, aminotransferases and ALP in early phase atherosclerosis, compared with the control group.

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by Novartis India, Mumbai, India) was administered subcutaneously at a dosage of 300 Ag/day/rat for 7 days. At the end of the 2 week experimental period, blood samples were collected and serum was separated for biochemical estimations and enzyme assays. Rats were sacrificed by decapitation; the heart, liver and kidney tissues were quickly excised, washed with saline, blotted with a piece of filter paper and a 10% (w/v) buffered homogenate was prepared for biochemical assays. Sections of heart, liver and kidney tissues were set aside for histologic processing. Experimental animals were used after obtaining prior permission and handled according to the University and institutional legislation, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

ical and histological changes in atherosclerotic cardiovascular disease have been investigated, and the role of LMWH has been traced through these events, in the cardiac, hepatic and renal tissues.

2. Materials and methods 2.1. Experimental protocol Adult male albino rats of Wistar strain (140 F 10 g) were housed in spacious cages and given food and water ad libitum. Animals were housed under standard conditions of temperature (23 F 1 jC), relative humidity (55 F 10%), 12-h light/12-h dark cycle. They were divided into four groups of six rats each. Group I served as the control. Rats in groups II and IV were fed an atherogenic diet comprising of the normal rat chow supplemented with 4% cholesterol, 1% cholic acid and 0.5% thiouracil (CCT diet) for 2 weeks; however, group IV also received LMWH treatment commencing 1 week after the start of the experimental period. Group III rats received LMWH alone. LMWH (Certoparin Sodium-TROPARIN; Mfd by: Biochemie GmBH, Austria, and marketed

2.2. Biochemical estimations in blood Serum albumin was assayed by the method of Reinhold [4]. Serum urea [5], uric acid [6] and creatinine [7] were estimated by standard procedures.

Table 2 Alterations in tissue enzyme activities in early phase atherosclerosis and the effect of LMWH treatment, compared with the control groups Enzyme assays (U/mg protein)

Group I Control

Group II CCT diet

Group III Control + LMWH

Group IV CCT diet + LMWH

Heart LDH AST ALT ALP

31.32 F 3.50 0.17 F 0.02 0.06 F 0.01 0.09 F 0.01

62.07 F 7.00a * 0.34 F 0.03a * 0.18 F 0.04a * 0.13 F 0.02a #

31.94 F 3.50 0.16 F 0.02 0.06 F 0.01 0.08 F 0.02

36.38 F 3.30b * 0.23 F 0.02a @b * 0.09 F 0.02b * 0.09 F 0.01b #

Liver LDH AST ALT ALP

11.20 F 1.01 0.14 F 0.01 0.11 F 0.01 1.45 F 0.15

16.03 F 1.45a 0.23 F 0.02a 0.27 F 0.03a 3.27 F 0.33a

* * * *

11.38 F 1.14 0.13 F 0.01 0.11 F 0.02 1.41 F 0.16

12.30 F 1.30b # 0.13 F 0.02b * 0.15 F 0.02a @ 1.77 F 0.18b *

13.50 F 1.50a # 0.20 F 0.02a # 0.52 F 0.04a * 1.90 F 0.10a *

9.65 F 0.97 0.12 F 0.01 0.27 F 0.02 1.30 F 0.12

9.46 F 0.85b # 0.14 F 0.02b @ 0.29 F 0.02b * 1.51 F 0.14b #

Kidney LDH AST ALT ALP

9.38 F 1.03 0.12 F 0.02 0.26 F 0.03 1.31 F 0.15

b

*

Values are expressed as mean F S.D. for six animals in each group. Enzyme units: LDH—Amol  10 1 of pyruvate liberated/min; AST,ALT— Amol  10 2 of pyruvate liberated/min; ALP—Amol  10 2 of phenol liberated/min. Statistical comparisons followed are as in Table 1. * p < 0.001. @ p < 0.05. # p < 0.01.

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2.3. Enzymic indices of cellular integrity Creatine kinase (CK) was assayed in the serum and heart tissue by the method of Okinaka et al. [8]. Activities of lactate dehydrogenase (LDH), aminotransferases (aspartate and alanine transaminases, AST and ALT, respectively) were assayed in serum and tissue (heart, liver and kidney); alkaline phosphatase (ALP) activity in the serum as well as cardiac, hepatic and renal tissue was assayed using disodium phenyl phosphate as substrate [9a,b,c]. 2.4. Analysis of serum lipids Serum total cholesterol and triglyceride were estimated by colorimetric methods [10,11]. The atherogenic index was expressed as LDL-c + VLDL-c/HDLc, where the lipoproteins were fractionated by a dual precipitation technique of Spiger [12]. After the fractional precipitation, the lipoprotein cholesterol was estimated [10]. 2.5. Histopathologic studies A portion of the heart, liver and kidney tissue, immediately after sacrifice was fixed in 10% formalin.

The washed tissue was dehydrated in the descending grades of isopropanol and finally cleared in xylene. The tissue was then embedded in molten paraffin wax. Sections were cut at 5 Am thickness, stained with haematoxylin and eosin. The sections were then viewed under light microscope for histopathological changes. 2.6. Statistical analysis The results were expressed as mean values F S.D. Differences between groups were assessed by ANOVA using the SPSS system for Windows.

3. Results In the present study, CCT diet feeding for 2 weeks was chosen as the experimental model of early phase atherogenesis, where cardioprotection rendered by the LMWH, certoparin sodium, was studied on the basis of its modulation of some biochemical variables, enzymic indices and histologic findings. The role of LMWH in combating the hepatic and renal aberrations accompanying diet-induced hypercholesterolemia have also been investigated here.

Fig. 2. The activity of CK enzyme in serum and heart tissue of atherogenic diet fed rats and the influence of LMWH treatment. Units: Serum— Amoles  10 3 of phosphorus liberated min 1; Heart—Amol  10 1 of phosphorus liberated min 1.

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Fig. 3. Serum cholesterol (shaded bars) and triglyceride (TG) concentrations in the untreated CCT diet fed rats and LMWH treated group.

Table 1 presents the changes in serum biochemical variables in the atherogenic diet fed rats, and the protective role of LMWH. Serum uric acid rises 1.88-fold in group II as against the control values, while its concentrations are in the normal range in the LMWH treated group IV. Lowered serum albumin concentrations in the CCT diet fed rats ( p < 0.001) may imply hepatic damage as well as abnormal renal glomerular function as a result of severe hyperlipidemia induced by the diet. Further, increased serum urea and creatinine concentrations ( p < 0.05 and p < 0.01, respectively) may serve to indicate developing glomerulopathy. Group IV shows the concentrations of these parameters to be comparable with the control group. Fig. 1 reveals the abnormal concentrations of serum enzymes that indicate cellular damage caused by the atherogenic diet. A 1.96-, 2.13-, 1.98-, and

1.59-fold increase in serum LDH, AST, ALT and ALP is recorded in group II as against the controls; however, LMWH treatment reverted the values to normalcy (group IV, p < 0.001). Table 2 displays the cardiac, hepatic and renal activities of LDH, AST, ALT and ALP. In the experimental model of early phase atherogenic fatty changes, the above enzymes exhibit significant rise in their activities ( p < 0.01, p < 0.001). Normalized tissue enzyme activity is observed in the LMWH treated CCT diet fed rats. The safety of the present dosage of LMWH is appreciated in group III, where enzyme activities remain in control range. Fig. 2 highlights cardiac injury in the atherogenic rats and the protective effect of LMWH treatment. Group II suffers 2.48-fold rise in serum CK concentrations and 53.57% diminished CK activity in the cardiac tissue, in comparison with the controls.

Table 3 Assessment of the atherogenic index (LDL-c + VLDL-c/HDL-c) in the untreated hypercholesterolemic group and the treated group Atherogenic index AI

Group I Control 0.98 F 0.07

Group II CCT diet a

4.76 F 0.34 *

Group III Control + LMWH

Group IV CCT diet + LMWH

0.95 F 0.07

1.09 F 0.08b *

Values are expressed as mean F S.D. for six animals in each group. Statistical comparisons and symbols followed are as in Table 1.

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Fig. 4. Histopathology of heart, liver and kidney in treated and untreated rats. (a) CCT diet fed group (H and E,  100), presents cardiac muscle hypertrophy. (b) CCT diet/LMWH treated group, (H and E,  100), absence of cardiac muscle hypertrophy, near normal histology. (c) CCT diet fed group (H and E,  400), reveals severe fatty changes in the liver cells. (d) CCT diet/LMWH treated group, (H and E,  100), normal parenchymal structure with very occasional fat cells, presents almost normal hepatocytes. (e) CCT diet fed group (H and E,  400), shows mild renal tubular damage. (f) CCT diet/LMWH treated group, (H and E,  100), presents normal renal histology.

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LMWH affords cardioprotection to CCT diet fed rats in group IV; serum and cardiac CK activities are reverted to normlacy ( p < 0.001). Fig. 3 shows the abnormally increased serum cholesterol and triglyceride concentrations as well as atherogenic index in the CCT diet fed rats, and the normalizing effect of LMWH. The rise in serum cholesterol is about three-fold when compared with the control values. Cholesterol concentration in LMWH treated group IV was significantly reduced compared to the untreated group II ( p < 0.001). The increased serum triglyceride concentration in group II ( p < 0.001) returned to normal by LMWH treatment ( p < 0.001). Nearly five-fold rise in atherogenic index (Table 3) was observed in the hypercholesterolemic rats (group II). The anti-atherogenic effect of LMWH is implicated by the reduced AI in group IV ( p < 0.001). The abnormal cardiac histology in response to the hypercholesterolemic diet (group II) is presented in Fig. 4a. The photomicrograph (H and E,  100) shows thickening of cardiac muscle cells and the onset of cardiac muscle hypertrophy. Fig. 4b (H and E,  100) depicts a more or less normal cellular architecture of the heart muscle cells, with notable absence of cardiac muscle hypertrophy in the LMWH treated CCT diet fed group IV. Fig. 4c (H and E,  400) show the fatty changes induced by the atherogenic diet in group II. The photomicrograph points out vacuolated hepatocytes with the nucleus being pushed to the periphery, and fatty cyst; an overall picture of fatty liver. Fig. 4d depicts a more or less normal hepatic architecture, with the parenchymal structure preserved and occasional fat cells in the LMWH treated CCT diet fed group. Fig. 4e shows mild tubular damage in the renal tissue (H and E,  400) corresponding to the untreated atherogenic diet fed group II, while Fig. 4f presents normal renal histology corresponding to the LMWH treated group IV.

4. Discussion Atherosclerosis is a slowly progressing, inflammatory, proliferative disease in which various cells such as macrophages, endothelial and smooth muscle cells (SMC) are involved [13]. Each character-

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istic lesion of atherosclerosis represents a different stage in a chronic inflammatory process in the artery; if unabated and excessive, this process will result in an advanced, complicated lesion. Rats maintained on CCT atherogenic diet for 2 weeks has been reported to result in significant mononuclear cell adhesion to the vessel wall, followed by their emigration into the intima where they accumulate lipid and become foam cells [14]. In the present study, we observed substantial protection rendered by LMWH against the cardiac, hepatic and renal changes in early stages of atherogenesis. It has been suggested that diet-induced hypercholesterolemia leads to changes in both the endothelium and circulating monocytes that precipitate these early events of lesion formation [15]. Hypercholesterolemia has been reported to cause endothelial cell dysfunction, as evidenced by an increase in endothelial cell turnover in cholesterol fed rabbits and swine [16,17] and increased permeability of the endothelium in cholesterol-fed rabbits [18]. The concept that lipids may participate in the progression of renal disease was first suggested over 100 years ago [19]. In recent times, there has been renewed interest in the toxic potential of lipids for both the glomerulus and tubules of the kidney. It has been noted that the hallmark histological lesion in the kidney in chronic renal failure, namely glomerulosclerosis, has many analogies to atherosclerosis. There is experimental evidence that an increase in dietary cholesterol favours the development of glomerulosclerosis in rats [3]. The glomerulus has many structural features that resemble arteries commonly involved in atherosclerosis. Glomerular mesangial cells are structurally similar to arterial smooth muscle cells, known to be important in the pathogenesis of atherosclerosis. Lipid-laden macrophages, or foam cells, frequently found in early atherosclerotic lesions are also found in glomeruli of human and experimental focal glomerulosclerosis [20]. These inflammatory cells are an early and significant component in the initiation of atherosclerosis. Hyperlipidemia may also induce vascular smooth muscle and mesangial cell proliferation [21]. Heparin is reported to safeguard against the above mentioned attributes of atherogenesis [22]. Exogenous heparin corrects the depletion of normal negative charge of the endothelial surface in vascular injury,

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and thereby restores normal function. Depletion of heparin-like anionic molecules predisposes to the development of atherosclerosis, because such areas have an increased susceptibility to endothelial injury, increased permeability, and an enhanced infiltration of cholesterol [23]. Numerous anti-inflammatory actions of heparin substantiate its protective role in atherosclerosis, where many inflammatory factors are involved in all stages of this process [24 – 26]. Vascular smooth muscle cell proliferation and migration are fundamental processes, and their inhibition by heparin has been reviewed [24,25]. With these multi-faceted anti-atherogenic properties of heparin in the backdrop, the results of the present study may be interpreted. The atherogenic diet fed rats in our study showed significant rise in serum uric acid concentrations, which was reverted to normalcy by LMWH treatment. Hyperuricemia has been reported to be an independent predictor of cardiovascular risk [27]. In another study, it was stated that hyperuricemia is a poor renal prognostic factor [28]. One of the mechanisms by which hyperuricemia may lead to poor renal prognosis is the linkage between hyperuricemia and inflammatory response. Another mechanism is the relationship between hyperuricemia and atherosclerosis. Increased oxidant stress and oxidation of low density lipoprotein (LDL) in arterial wall by peroxynitrite play an important role in the development of atherosclerosis. In this sense, the finding of elevated serum uric acid concentrations may reflect the body’s response to an increased production of endogenous oxygen species because uric acid is a potent scavenger of peroxynitrite [29]. Suarna et al. [30] reported a more direct role of uric acid in the progression of the atherosclerotic process, because urate crystals trigger an inflammatory response in the vascular plaque. The decrease in serum albumin in atherogenic diet fed rats may be due to its increased urinary excretion due to glomerular barrier abnormality as well as the increased permeability of vascular tissue to albumin via increasing the concentration of cAMP by inflammatory mediators [31]. The increase in albumin concentrations by LMWH may therefore be due to restoration of normal glomerular function and decrease of albumin extravasation occurring in response to inflammation. In a study by Stefoni et al. [32], the serum albumin concentrations were found to be significantly lower in hemodialysis patients with cardio-

vascular disease, wherein albumin was implicated as an independent risk factor for cardiovascular death. In their study serum albumin showed a direct correlation with serum TGF-h1 concentrations in hemodialysis patients with cardiovascular disease and both the parameters seemed to reflect the severity of vascular impairment and/or the degree of vascular inflammation. The hypoalbuminemic condition in our CCT diet fed rats implicates the onset of cardiovascular disease. LMWH treatment restores normal serum albumin concentrations, thereby projecting its cardiac and renal vascular protective role. Enzymic markers of cellular damage with concomitant inflammation showed sharp increases in the serum and tissue activities of LDH, AST, ALT and ALP in the diet induced group; the anti-inflammatory properties of the heparin-derivative restores normalcy. Increases in tissue enzyme activities have been reported in experimental inflammatory conditions [33]. Phosphatase activity on endothelial cell surfaces is responsible, in part, for the conversion of adenosine nucleotides to adenosine, a potent vasodilator and anti-inflammatory mediator that can protect tissues from the ischemic damage that results from injury. Therefore, following injury, accumulation of interleukin-6 can lead to production by alkaline phosphatase of adenosine and subsequent protection from ischemic injury [34]. Such an induction of ALP activity may substantiate the increased activity of this enzyme in the atherogenic group. Hypercholesterolemia-induced microvascular alterations characteristic of oxidative stress and an inflammatory response have been reported in animals within a few days of feeding a cholesterol enriched diet, i.e., long before the appearance of fatty streak lesions in large arteries [35,36]. It is generally assumed that hypercholesterolemia increases the incidence of ischemic disease in tissues (such as heart and brain) by restricting blood flow in lesion-prone arteries. However, in a recent review [37], there is convincing evidence indicating that hypercholesterolemia may also promote ischemic tissue damage by enhancing the vulnerability of the microcirculation to the deleterious effects of ischemia and other inflammatory stimuli. In the light of these facts, LMWH proves to be extremely useful as it counters the lipemic disturbances, inflammatory events and oxidative injury,

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thereby protecting the tissues against hypercholesterolemia-induced deleterious changes. Hyperlipidemia which is a risk factor for atherosclerosis is substantiated by our finding of significantly elevated blood cholesterol and triglyceride concentrations and a high atherogenic index in the CCT diet fed rats. The treated rats show a decline in these parameters towards normalcy, indicating that the heparin derivative tested in this study, certoparin, to exert anti-lipemic and anti-atherogenic roles. Engelberg [38] has comprehensively reviewed the antiatherogenic effect of heparin, wherein a deficiency of endogenous heparin activity is said to result in hypertriglyceridemia, hypercholesterolemia and enhanced atherogenesis, which is corrected on injecting exogenous heparin. Low concentrations of high-density lipoproteins are an independent risk factor for atherosclerosis, however, when heparin is injected, there is an absolute increase in high-density lipoproteins [22]. In the present study, the heparin-derivative treated group showed favourable modulation of lipoproteins, wherein the increased HDL and lowered LDL, VLDL accounted for the reduced atherogenic index, while the untreated hypercholesterolemic group had a high atherogenic index on account of its decreased HDL and increased LDL, VLDL concentrations. LMWH treatment possesses the additional merit that it has weaker plasma lipolytic potential than heparin. As heparin induces a rapid lipolytic process leading to substantial rises in plasma concentrations of free fatty acids and partial glycerides, which on high concentrations may affect cardiac function adversely, LMWH administration may be preferred in clinical situation [39]. Lipemia impedes oxygen diffusion thereby adversely affecting tissue oxygenation, and hypoxia, which in turn accelerates the atherosclerotic process [40]. The increased cholesterol content in the red blood cells of hypercholesterolemic animals occurs primarily in the cell membrane where it may serve as a barrier to oxygen transfer [41]. The decrease in lipemia after heparin injection increases tissue oxygen supply [42]. This property accounts for heparin’s protective role in atherogenesis and in tissue and myocardial function [38]. We have earlier reported that LMWH administration decreases membrane cholesterol and lipid peroxidation in conditions of primary and secondary hypercholesterolemia, where a

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significant elevation in these two parameters was observed [43]. In the present work, we demonstrated the tissue protective properties of LMWH in hypercholesterolemic conditions. In lipemic conditions, we earlier observed accentuated oxidative stress in the cardiac, hepatic and renal tissues, which was normalized by LMWH treatment [44]. Campos et al. [45] have demonstrated that hypercholesterolemia predisposes renal tubular cells to hypoxic injury, wherein hypercholesterolemia mediates a direct effect on epithelial tubular cells, which become more susceptible to ischemic injury. The mild renal lesion seen in the histology figure corresponding to the untreated hypercholesterolemic diet fed group in the present study may be attributed to the lipemic oxidative injury. The latter is offset by LMWH administration that accounts for the normal renal histology in the LMWH treated rats. Besides the above discussed biochemical evidence favoring the protective role of the low molecular weight heparin fragment, certoparin, in countering the cellular injury induced by the hypercholesterolemic atherogenic diet, we also indicate the anti-hypertrophic effect of the heparin derivative. Histologic examination of the cardiac tissue of untreated atherogenic rats revealed mild hypertrophy of the cardiac muscle cells. An earlier report strongly suggested that heparin and heparan sulfate are potent inhibitors of cardiomyocyte hypertrophy and that endogenous heparin-like substances negatively regulate cardiomyocyte hypertrophy [46]. In our study, the treated rats exhibited almost normal cardiac histology, indicating an anti-hypertrophic effect exerted by the low molecular heparin fragment, certoparin. The lipid-lowering effect of LMWH protected the liver cells against fatty changes.

5. Conclusions The study showed that certoparin sodium exerts a protective effect on the early detrimental changes in the heart, liver and kidneys, consequent to atherogenic diet feeding. On histologic examination, the onset of a mild cardiac muscle hypertrophy is indicated in the atherogenic diet fed rats, while LMWH treatment restores normal cardiac architecture. Endothelium protection by replenishing the negative charges lost due to injury, anti-inflammatory property, and inhibition of hyper-

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plasia and hypertrophy by heparins, substantiate the salubrious role of the heparin-derivative, certoparin in mitigating the cardiac, hepatic and renal abnormalities arising due to the major atherosclerotic risk factor, hypercholesterolemia.

Acknowledgements We thank Dr. C.S.Vijayalakshmi, M.D. (Path), D.C.P., for her valuable help in interpreting the histological specimens. The Junior Research Fellowship awarded to the first author, Deepa P.R., by the Indian Council of Medical Research (ICMR), New Delhi (India), is gratefully acknowledged.

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