Lipid profile and histological measurements of iliac artery in various experimental groups.

<p>Results expressed as mean ± SEM. TC: Total Cholesterol, TG: Triglyceride, LDL: Low density lipoprotein, HDL: High density lipoprotein, I/M thickness ratio: Intimal/medial thickness ratio, %CSN: Percentage cross sectional narrowing. ***p<0.001 vs. normal; #p<0.05, ##p<0.01 and ###p<0.001 <i>vs</i> Baseline. number of sections = 25–30 sections/group.</p

Cholesterol Diet Withdrawal Leads to an Initial Plaque Instability and Subsequent Regression of Accelerated Iliac Artery Atherosclerosis in Rabbits

<div><p>Effect of long term cholesterol diet withdrawal on accelerated atherosclerosis in iliac artery of New Zealand White (NZW) rabbits has not been explored so far. Atherosclerosis was thus induced in rabbits by a combination of balloon injury and atherogenic diet (AD) (1% cholesterol and 6% peanut oil) feeding for 8 weeks (baseline) followed by chow diet (CD) feeding for 4, 8, 16, 32, 50 and 64 weeks. The plaque characterization was done using histology, real time RT-PCR and vasoreactivity studies. Significant elevation in plasma lipids with AD feeding was normalized following 16 weeks of CD feeding. However, baseline comparison showed advanced plaque features even after 8 weeks of CD period with significant elevation in intima/media thickness ratio and plaque area later showing reduction at 50 and 64 weeks CD periods. Lesion lipid accumulation and CD68 positivity was maintained till 16 weeks of CD feeding which significantly reduced from 32 to 64 weeks CD periods. Baseline comparison showed significant increase in ground substance, MMP-9 and significant decrease in α-actin and collagen content at 8 weeks CD period indicating features of unstable plaque. These features regressed up to 64 weeks of CD. Partial restoration of functional vasoconstriction and vasorelaxation was seen after 64 weeks of CD feeding. mRNA expression of MCP-1, VCAM-1, collagen type I and III, MMP-9, TIMP-1, IFN-γ, TNF-α, IL-10 and eNOS supported the above findings. The study thus reveals insights into initial plaque instability and subsequent regression on AD withdrawal in this model. These results are suggestive of an appropriate window for drug intervention for plaque stability/regression and restenosis as well as improves understanding of plaque regression phenomenon in this model.</p></div

Vasomodulatory effects and mass spectral analysis of <em>Bridelia ferruginea</em> Benth.

621-628Bridelia ferruginea Benth. (Fam. 'Euphorbiaceae) is known to possess potent anti-inflammatory activity. Here, we investigated its vasomodulatory effect, as anti-inflammatory therapy that beneficially impact the cardiovascular system. Extracts (Bf1, Bf-HA) and fraction (Bf2) of B. ferruginea (Bf), were prepared from the bark of Bf to study their vasomodulatory effect using rat aortic rings. The vasorelaxant effect of Bf1 and Bf2 was mediated by the activation of nitric oxide synthase/endothelial isoform (NOS3) as confirmed by EA.hy926 endothelial cells, real-time PCR and Western blotting. Mass spectral analysis of these extracts and fraction was performed to understand the profile of compounds present in them. Mass spectral analysis showed the presence of similar ions in both Bf1 and Bf2 while Bf-HA showed different patterns. Vasorelaxant effect of Bf1 and Bf2 in phenylephrine (PE) pre-contracted endothelium intact aortic rings was blocked significantly in the presence of both N-nitro-L-arginine methyl ester (L-NAME) or soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one [ODQ]). However, cyclo-oxygenase (COX) inhibitor (indomethacin) did not exert any change. In contrast, Bf-HA significantly inhibited ACh-induced vasorelaxation, but had no effect on sodium nitroprusside (SNP)-mediated relaxation, thereby suggesting NOS inhibitory activity in the extract. Studies with Bf1 and Bf2 on EA.hy926 cells demonstrated NOS3 mediated nitric oxide (NO) generation. Purified fractions of Bf, thus possess vasorelaxant compounds, which remain to be identified

Biphasic elevation and subsequent regression of ground substance and matrix metalloproteinase-9.

<p>Panel (A) Representative images of movat pentachrome stained sections of all groups (Scale bar  = 50 µm). Panel (B) Immunohistochemical staining showing MMP-9 positive area of all groups (Scale bar  = 50 µm). (C) Quantitative analysis of alcian blue stained area in respective groups (D) Quantitative analysis of MMP-9 positive areas in respective groups (E) Arterial MMP-9 mRNA expression (fold change over normal) in all groups as determined by real time PCR (F) Arterial TIMP-1 mRNA expression (fold change over normal) in all groups as determined by real time PCR. **p<0.01 and ***p<0.001 <i>vs</i> normal; ^#p<0.05, ^##p<0.01 and ^###p<0.001 <i>vs</i> Baseline. (* indicates lumen, arrow heads indicate positive staining)</p

Vasoreactivity studies of injured iliac artery of all groups.

<p>(A) Effect of incremental doses (1 nM–100 µM) of phenylephrine induced contractions on atherosclerotic iliac arteries of respective groups (B) Effect of incremental doses (3 nM–3 mM) of acetylcholine induced relaxation on atherosclerotic iliac arteries of respective groups (C) Arterial eNOS mRNA expression in all groups as determined by real time PCR. **p<0.01 and ***p<0.001 <i>vs</i> normal; ^#p<0.05, ^##p<0.01 and ^###p<0.001 <i>vs</i> Baseline.</p

Regression phase leads to progressive decrease in percentage lipid area and macrophage foam cell content.

<p>Panel (A) Representative images of hematoxylin and eosin stained sections of all groups (Scale bar  = 500 µm). Panel (B) Representative images of Oil Red O stained cryostat sections of all groups (Scale bar  =  50 µm). Panel (C) Immunohistochemical staining showing CD68 positive cells in respective groups. (Scale bar  = 50 µm) (D) Quantitative analysis of percentage lipid area (lumen area included) in respective groups (E) Quantitative analysis of CD68 positive labelling in respective groups. **p<0.01 and ***p<0.001 <i>vs</i> normal; ^##p<0.01 and ^###p<0.001 <i>vs</i> Baseline. (* indicates lumen, arrow heads indicate positive staining)</p

Experimental Protocol.

<p>Sixty four male New Zealand White rabbits were fed atherogenic diet (AD) for 8 weeks to create atheroma. Seven animals were fed chow diet (CD) for 8 weeks. Balloon injury of iliac artery by Fogarty embolectomy catheter was performed 1 week after initiation of atherogenic diet. Baseline group was set at 8 weeks of atherogenic diet feedingfollowed by chow diet (CD) from 4 weeks up to 64 weeks in respective groups. n = number of animals</p

Collagen (Sirius red staining) and α-actin smooth muscle content in various groups.

<p>Panel (A) Representative images of Siruis red stained section of all groups under polarized light. Brightfield image of same section is shown as an inset (Scale bar = 50 µm). Panel (B) Immunohistochemical staining of α-actin in sections of all groups (Scale bar = 50 µm). (C) Quantitative analysis of α-actin positive stained area in respective groups (D) Arterial collagen I mRNA expression (fold change over normal) in all groups as determined by real time PCR (E) Arterial collagen III mRNA expression (fold change over normal) in all groups as determined by real time PCR. ***p<0.001 <i>vs</i> normal; ^###p<0.001 <i>vs</i> Baseline. N.D =  not detectable (* indicates lumen, arrow heads indicate positive staining)</p