Drag-Reducing Polymers Improve Left Ventricular Hypertrophy And Aortic Remodeling In Spontaneous Hypertensive Rats

Background and Significance:In both human and animal models, hypertension has been associated with cardiac and aortic remodeling.Cardiac remodeling is defined as genome expression resulting in molecular, cellular and interstitial changes and manifested clinically as myocardial hypertrophy, fibrosis and cardiac decompensation. Aortic remodeling is characterized by impaired endothelium-dependent vasodilation and structural vascular changes and the endothelium plays a primary role in the modulation of vascular tone and structure.The process of cardiac remodeling is influenced by hemodynamic load, neurohormonal activation and other factors still under investigation. The entire cardiovascular system is lined by a thin layer of cells known as the vascular endothelium which directly contacts flowing blood and transduce hemodynamic stimuli into changes in endothelial structure and function. Hemodynamic shear stress from blood flow acting on the endothelium critically regulates vascular morphogenesis and blood pressure.What’s more, a rapid and significant down-regulation of endothelin 1 (ET-1)mRNA and peptide release in bovine aortic endothelial cells have be observed under hemodynamic shear stress.Drag-reducing polymers (DRP) are long-chain, blood soluble macromolecules, have been shown to greatly reduce frictional resistance in flow. Previous studies have demonstrated that intravenous injection of even nanomolar concentration of DRP increased hemodynamic shear stress and decreased peripheral vascular resistance.In this study,we hypothesis that the intravenous infusion of DRP may improve left ventricular hypertrophy and aortic remodeling in spontaneous hypertensive rats(SHR).Subjects and Methods:1. Preparation of DRPDRP solution was prepared by polyethylene oxide (PEO,Sigma-Aldrich,Saint Louis, MO), with an average molecular weight (MW) of 5×106 Da. The polymer was carefully dissolved in normal saline at a concentration of 1000 ppm, avoiding mechanical shear degradation of the long-chain macromolecules. And then dialyzed against normal saline for 24 hours using a membrane(Regenerated Cellulose Dialysis Membrane, Spectra, Spectrum Laboratories Inc.) with 50-kDa MW cutoff. The stock PEO solution of 1000 ppm concentration was diluted to a concentration of 20 ppm or 10ppm with saline and mixed for 1 hour on a slow rocker prior to use.2. Animals and protocolsAll experimental protocols in this study were approved by the Animal Care and Use Committee of the Southern Medical University (Guangzhou, Guangdong, China) and were in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no.85-23, revised 1996).24 SHR s(age,8 weeks; weight,180-220 g, provided by the Vital River) were randomly divided into three groups:normal saline group (n=8),10 ppm group (n=8),20 ppm group (n=8). One control group of age matched normotensive Wistar-Kyoto (WKY) rats received normal saline. A syringe pump (TE311, Terumo Corperation, Tokyo) was utilized to guarantee a constant-rate infusion at 3 ml/h 30 min via the rat tail vein every two days for two months. Rats were housed at constant room temperature, humidity, and light cycles (12/12h light-dark), had free access to tap water, and were fed standard chow ad libitum.3. Measurement of body weight(BW), heart rate(HR) and systolic blood pressure (SBP)BW,HR and SBP was measured at the beginning,20th,40th and 60th days of the experiment when all animals were in conscious conditions.SBP was measured in all rats using tail-cuff method 17.Ambient temperature was maintained at 30℃. Before the measurement, the rats were trained to adapt themselves to the restraining cages and tail-cuff apparatus for the standard non-invasive tail-cuff.4. Echocardiography measurementTransthoracic echocardiography was performed with the use of a high resolution echocardiographic system (Sequoia512, Acuson, Siemens) equipped with a 14-MHz linear transducer (17L5 probe, Acuson, USA). The transducer was located overlying the chest to produce the optimal parasternal short axis two-dimensional images. A parasternal short-axis M-mode image at the papillary muscle level was acquired to measure the left ventricular end-systolic diameter (LVESD) and -diastolic diameter (LVEDD), left ventricular end-systolic posterior wall thickness (LVPWS). Left ventricular systolic function was also assessed from these measurements by calculating the LV fractional shortening (LVFS) and the LV ejection fraction (LVEF).5. Measurement of plasma and myocardial ET levelBoth plasma and homogenated myocardial ET levels were measured by using an ELISA kit (Biocalvin, Catalog No.EIA-3600,Shanghai, China) according to the manufacturer’s instructions.6. Histological assessment of thoracic aortaThoracic aorta was collected and fixed in 10% formalin, dehydrated, and then embedded in paraffin. Subsequently,4μm thick sections were cut and stained with hematoxylin eosin statin. The medial thickness of thoracic aorta was quantified from each group. The expression of ET-1 in thoracic aorta was detected by Immunohistochemistry.All image analysis was performed in a blind manner using Image Pro Plus (version 6.0, Media Cybernatics,USA).7. Histological assessment of left ventricleThe left ventricle was collected and fixed in 10% formalin, dehydrated, and then embedded in paraffin. Subsequently,4μm thick sections were cut and stained with hematoxylin eosin statin and Masson’s trichrome stain. The cross-sectional areas of cardiomyocytes and collagen volume fraction in left ventricle were quantified from each group. The expression of ET-1 of left ventricle was examined by immunohistochemistry. The expression of ET-1 mRNA of left ventricle was detected by qRT-PCR.Result:1.Body weight, heart rate and systolic blood pressuresDuring 60 days of treatment, body weight increased over time and there were no statistically significant differences among the SHR groups as shown in Fig.1. The SBP in WKY+NS were significantly lower than that in all SHR groups during the treatment since it increased steady and smoothly while there was no significant difference between the SHR groups. Heart rate did not differ among all groups.2. DRP improve left ventricular posterior wall hypertrophyAt the 60th days, M-mode echocardiography was conducted in vitro to prove whether or not myocardium of SHR underwent hypertrophy. The results indicated an increase in LVPWS(3.31±0.21 vs 2.62±0.11,p<0.05) and LVPWD(2.58±0.05 vs 1.91±0.11,p<0.05) of SHR+NS compared with WR+NS. But after DRP administration,the LVPWS of SHR+10 and SHR+20 was significantly decreased compared with SHR+NS (2.65±0.12 and 2.63±0.14 vs 3.31±0.21.P< 0.05, respectively).What’s more, the LVPWD of SHR+20 was significantly decreased compared with SHR+NS(2.03±0.14 vs 2.58±0.05,P<0.05) (Fig.2).Besides, LVESD and LVEDD of SHR+10 and SHR+20 were significantly increased compared with SHR+NS,but there was no significant difference in FS and EF.3.DRP improve left ventricular hypertrophy and myocardium fibrosisThere was an increase in cross-sectional area of SHR+NS compared with that of WKY+NS(630.97±54.15μm2 vs 309.34±9.9μm2,P< 0.05) (Fig.3). These changes were attenuated by DRP treatment, as the f cross-sectional area of SHR+20 was significantly suppressed (291.72±29.08μm2 vs 630.97±54.15μm2,P=0.016). Masson’s trichrome staining clearly showed increased collagen volume fraction in the left ventricle(LVCVF) in SHR+NS compared with WR+NS (6.58 ±0.49% vs 1.86 ±0.37%,P=0.000).These changes were significantly reversed in SHR+10 and SHR+20 groups (2.70±0.34%,2.38±0.34% vs 6.58±0.49%,P< 0.05, respectively), but without significant differences between them。4. DRP improve aortic medial thicknessThe medial thickness of aorta in SHR+NS was significantly higher than that of WR+NS(164.97±3.37μm vs 126.63±4.0μm,P=0.002),The treatment with DRP significantly reduced the medial thickness in SHR+10 and SHR+20(132.73± 7.12μm,135.46±6.79μm vs 164.97±3.37μm,P< 0.05), thus showing an attenuation of vascular hypertrophy.5. DRP attenuated ET-1 expression in LVWe confirmed that both ET-1 gene and protein expression were attenuated by DRP administration.Immunohistochemistry results clearly showed increased expression of ET-1 protein in the cardiomyocytes of SHR+NS than that of WR+NS,but attenuated by DRP administration in SHR+10 and SHR+20. Real-time PCR showed that ET-1 gene expression was markedly decreased in SHR+20 than that of SHR+NS (1.46±0.14 vs 2.24±0.07, P=0.001)6. DRP attenuated ET-1 expression in thoracic aortaImmunohistochemistry results clearly showed increased expression of ET-1 protein in the thoracic aorta of SHR+NS than that of WR+NS,but attenuated by DRP administration in SHR+10 and SHR+20.7. DRP attenuated ET-1 expression in serum and LV tissueCompared with the SHR±NS group, the levels of serum ET decreased in SHR+10 group and SHR+20 group (P<0.05) after DRP administration. And these results were in parallel to the left ventricular tissue ET levels of each group.Conclusion:These results suggest that chronic treatment with DRP can protect against left ventricular hypertrophy and aortic remodeling in spontaneous hypertensive rats. DRP may offer a new approach to the treatment of left ventricular hypertrophy and aortic remodeling caused by hypertension.